The proto-oncogene c-myc in hematopoietic development ... - Nature

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Oncogene (2002) 21, 3414 ± 3421 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc

The proto-oncogene c-myc in hematopoietic development and leukemogenesis Barbara Homan*,1, Arshad Amanullah1, Marianna Shafarenko1 and Dan A Liebermann1 1

Fels Institute for Cancer Research and Molecular Biology, Department of Biochemistry, Temple University School of Medicine, 3307 North Broad Street, Philadelphia, Pennsylvania, PA 19140, USA

The proto-oncogene c-myc has been shown to play a pivotal role in cell cycle regulation, metabolism, apoptosis, dierentiation, cell adhesion, and tumorigenesis, and participates in regulating hematopoietic homeostasis. It is a transcription regulator that is part of an extensive network of interacting factors. Most probably, dierent biological responses are elicited by dierent overlapping subsets of c-Myc target genes, both induced and suppressed. Results obtained from studies employing mouse models are consistent with the need for at least one, and possibly two, mutations in addition to deregulated c-myc for malignant tumor formation. Repression of c-myc is required for terminal dierentiation of many cell types, including hematopoietic cells. It has been shown that deregulated expression of c-myc in both M1 myeloid leukemic cells and normal myeloid cells derived from murine bone marrow, not only blocked terminal dierentiation and its associated growth arrest, but also induced apoptosis, which is dependent on the Fas/CD95 pathway. There is evidence to suggest that the CD95/Fas death receptor pathway is an integral part of the apoptotic response associated with the end of the normal terminal myeloid dierentiation program, and that deregulated c-myc expression can activate this signaling pathway prematurely. The ability of egr-1 to promote terminal myeloid dierentiation when coexpressed with c-myc, and of c-fos to partially abrogate the block imparted by deregulated c-myc on myeloid dierentiation, make these two genes candidate tumor suppressors. Several dierent transcription factors have been implicated in the down-regulation of c-myc expression during dierentiation, including C/EBPa, CTCF, BLIMP-1, and RFX1. Alterations in the expression and/or function of these transcription factors, or of the c-Myc and Max interacting proteins, such as MM-1 and Mxi1, can in¯uence the neoplastic process. Understanding how c-Myc controls cellular phenotypes, including the leukemic phenotype, should provide novel tools for designing drugs to promote dierentiation and/ or apoptosis of leukemic cells. Oncogene (2002) 21, 3414 ± 3421 DOI: 10.1038/sj/ onc/1205400

*Correspondence: B Homan; E-mail: ho[email protected]

Keywords: c-myc; hematopoiesis; dierentiation; leukemogenesis Introduction Hematopoiesis is a profound example of cell homeostasis that is regulated throughout life, whereby a hierarchy of hematopoietic progenitor cells in the bone marrow (BM) proliferate and terminally dierentiate along multiple, distinct cell lineages (Metcalf, 1989; Quesenberry, 1990; Sachs, 1987) Clearly, a variety of control mechanisms are needed to maintain steady state levels of mature blood cells, as well as to stimulate the rapid production of speci®c cell types as needed. To achieve this requires the participation of many factors, including positive and negative regulators of growth and dierentiation, which determine survival, growth stimulation, growth arrest, dierentiation, functional activation, and apoptosis (Metcalf, 1991; Selvakumaran et al., 1994). Lesions in genes that control blood cell homeostasis may lead to either the development of anemia, or blood cell hyperplasia, which may progress to preleukemia, and ultimately to leukemia resistant to cancer therapy (McKenna and Cotter, 1997). One of many genes that participates in regulating hematopoietic homeostasis is the proto-oncogene cmyc. C-Myc has been shown to play a pivotal role in the regulation of proliferation, dierentiation, and apoptosis (Homan and Liebermann, 1998; Askew et al., 1991; Evan and Littlewood, 1993; Shi et al., 1992) As expected for a gene product that participates in these dierent processes, alterations in c-myc expression are associated with hematological malignancies (Boxer and Dang, 2001; Nesbit et al., 1999). Therefore, it is important to understand how c-Myc carries out its multiple functions, both with regard to normal hematopoiesis, the establishment and maintenance of hematological malignancies, and the responsiveness of these leukemias to dierent therapeutic regimens. The gene product encoded by the c-myc oncogene is a transcription factor. The carboxy terminus of c-Myc contains three structural domains that are homologous to domains found in characterized transcription factors, including a leucine zipper (Zip), a helix ± loop ± helix motif (HLH), and an adjacent domain rich

c-myc in hematopoiesis and leukemia B Hoffman et al

in basic amino acids (b) (Blackwell et al., 1990; Landshulz et al., 1988; Murre et al., 1989). The HLH and Zip motifs promote protein ± protein interaction, and the basic region mediates sequence-speci®c DNA binding (Blackwell et al., 1990; Landshulz et al., 1988; Murre et al., 1989). These motifs are contiguous, thus c-Myc is a bHLHZip protein (Henriksson and Luscher, 1996). The amino terminus of c-Myc (aa 1 ± 143), includes Myc box I (MBI) and Myc box II (MBII), conserved regions in all Myc family members. Structure-function studies have revealed that the bHLHZip domain is necessary for transcription regulation; the entire N-terminus is required for ecient transcription transactivation, whereas only MBII appears to be necessary for transcription repression (Claassen and Hann, 1999; Henriksson and Luscher, 1996). Consistent with the predominant localization of c-Myc in the nucleus, there are two nuclear localization signals in the carboxy terminus (Henriksson and Luscher, 1996). The c-Myc network c-Myc, a transcription regulator, is part of a network of interacting factors (Sakamuro and Prendergast, 1999), and this network ultimately controls Myc function. Only a brief overview will be presented here; there are many review articles dealing with this subject matter (Sakamuro and Prendergast, 1999; Baudino and Cleveland, 2001). Under physiological conditions cMyc cannot form homodimers (Henriksson and Luscher, 1996). Max, a ubiquitous bHLHZip protein appears to be an obligate heterodimeric partner for cMyc in mediating its functions (Amati and Land, 1994; Ayer and Eisenman, 1993). Initially two partners for Max had been identi®ed, Mxi1 and Mad (Amati and Land, 1994). Analysis of mouse Mxi1 has led to the identi®cation of two transcript forms, Mxi-SR and Mxi-WR (Schreiber-Agus et al., 1995). Two additional Max-interacting proteins, Mad3 and Mad4, which behave similarly to Mad (Mad1), have been reported (Henriksson and Luscher, 1996; Hurlin et al., 1995). It has been proposed that transcription activation is mediated exclusively by Myc:Max complexes, whereas Max:Max, Max:Mad and Max:Mxi complexes mediate transcription repression through identical binding sites (Ayer and Eisenman, 1993; Ayer et al., 1993; Zervos et al., 1993). mSin3A and mSin3B, possessing sequence homology with the yeast transcription repressor Sin3, bind speci®cally to Mad and Mxi. Evidence is consistent with mSin3 forming a ternary complex with Mad-Max and Mxi1-Max that recognizes E box binding sites and participates in transcription repression (Ayer et al., 1995; Harper et al., 1996; Rao et al., 1996; Schreiber-Agus et al., 1995); this repression is a consequence of the association of Sin3 with multiprotein complexes including histone deacetylases which alter chromatin structure and block transcription (Alland et al., 1997; Hassig et al., 1997; Laherty et al., 1997). Another Max-interacting partner, MNT, has

been identi®ed, which is coexpressed with Myc yet mediates repression of expression (Hurlin et al., 1997). It has been established that Max is essential for cMyc-mediated gene transactivation, transformation, cell cycle progression and apoptosis (Amati and Land, 1994; Packham and Cleveland, 1995). Overexpression of max in transgenic mice (Eu-myc/Eu-max) also overexpressing c-myc reduced the rate of lymphoma onset compared to Eu-myc transgenic mice, suggesting that elevation of Max expression in vivo inhibits the function of c-Myc (Lindeman et al., 1995). Overexpression of mxi1 (or Mxi-SR) and mad can antagonize c-Myc activity in cellular transformation assays (Koshinen et al., 1995; Lahox et al., 1994) and proliferation (Roussel et al., 1996; Wu et al., 1996), and can diminish the malignant phenotype of tumor cells (Chen et al., 1995). In addition, overexpression of mad1 reversed the dierentiation blocking eect of cMyc in erythroleukemia cells (Cultraro et al., 1997). These ®ndings are consistent with the notion that Mad and Mxi1 suppress c-Myc function (Packham and Cleveland, 1995). In the last several years, a plethora of c-Mycinteracting factors have been identi®ed, which may account for the versatile functions of c-Myc. Besides Max, cellular proteins that interact with the carboxy terminal domain (CTD) include YY-1, AP-2, BRCA-1, TFII-I, Nmi, SNF5, cdr2, and Miz-1. The amino terminal domain (NTD) interacts with the retinoblastoma family protein p107, TBP, Bin-1, AMY-1, TRRAP, PAM, a-tubulin, MM-1 and p21 (Baudino and Cleveland, 2001; Elliott et al., 2000; McMahon et al., 2000; Mori et al., 1998; Sakamuro and Prendergast, 1999). TRRAP mediates recruitment of the histone acetyltransferase hGCN by c-Myc, providing a biochemical basis for the antagonistic eects of Myc and Mad (McMahon et al., 2000). Alterations in the expression or function of the dierent c-Myc interacting proteins can eect the function of c-Myc.

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Consequences of deregulated c-myc expression on dierentiation and viability Effect of deregulated expression of c-myc on myeloid differentiation c-myc is expressed in almost all proliferating normal cells, and its repression is required for terminal dierentiation of many cell types, including myeloid cells (Freytag, 1988; Homan-Liebermann and Liebermann, 1991; Homan et al., 1996). It has been shown that deregulated expression of c-myc in both M1 myeloid leukemic cells and normal myeloid cells derived from murine bone marrow not only blocked terminal dierentiation and its associated growth arrest, but also induced a p53-independent apoptotic pathway (Amanullah et al., 2000). The apoptotic response was not completely penetrant; both proliferation and apoptosis were continuously ongoing in the same population of cells. Oncogene


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Analysis of the molecular mechanisms involved in the c-Myc-mediated apoptotic response associated with blocks in myeloid dierentiation revealed that it is dependent on the Fas/CD95 pathway (Amanullah et al., 2002). It was shown that c-Myc is a transcription regulator of CD95L, consistent with other studies that have suggested that c-Myc is required for the regulation of CD95 ligand expression in the process of activation-induced cell death in T-cells (Amanullah et al., 2002; Brunner et al., 2000; Genestier et al., 1999). In combination with the myeloid cell dierentiation inducer interleukin-6 (IL-6), c-Myc suppressed RNA levels of c-FLIP, an inhibitor of the CD95/Fas pathway (Amanullah et al., 2002). That c-FLIP was not completely absent could account for the incomplete penetrance of the apoptotic response. The mechanism by which c-Myc (in collaboration with the dierentiation inducer IL-6) down regulates c-FLIP is not clear. The critical nature of c-FLIP downregulation in the apoptotic response is revealed by the observation that exogenous c-FLIP restored the normal growth kinetics of M1myc cells and completely rescued the apoptotic phenotype. Evidence was obtained that cell death following normal myeloid terminal dierentiation utilizes the CD95/Fas apoptotic pathway (Amanullah et al., 2002). The observations of Fecho et al (1998) with bone marrow cells from CD95-mutant and CD95L-mutant mice indicate that the absence of either CD95 or CD95L has only minor consequences for granulopoiesis; however, it was also reported that fas-de®cient marrow contained increased myeloid progenitor potential (Traver et al., 1998). It is interesting to note that constitutive expression of the apoptotic suppressor Bcl2 in myeloid cells of CD95-mutant Faslpr/lpr mice often leads to a fatal disease analogous to human acute myeloblastic leukemia (Traver et al., 1998). Since approximately 15% of the mice develop disease, most probably additional oncogenic events are involved in the progression to the leukemic state. Based on our ®ndings, deregulated c-myc expression could be such an event. This is also consistent with the reported observation that loss of Fas receptor accelerated lymphomagenesis in Eu L-myc transgenic mice (Zornig et al., 1995). In the c-Myc-mediated apoptosis associated with blocks in dierentiation of myeloid cells, both the Type I and Type II CD95/Fas pathways play a role; however, the Bcl-2-sensitive Type II pathway is initially activated. It has recently been shown that c-Myc can suppress anti-apoptotic Bcl-2 family members (Eischen et al., 2001), which suggests additional subtleties in the eect of c-Myc on apoptosis. Suppression of Bcl-2 or Bcl-XL by c-Myc may further potentiate the Type II CD95/Fas apoptotic pathway. Interestingly, Bcl-2 and/ or Bcl-XL levels are elevated in more than half of the lymphomas arising in Eu-myc transgenic mice (Eischen et al., 2001), revealing the lost ability of c-Myc to suppress these anti-apoptotic proteins during c-Myc induced tumorigenesis. After back crossing CD2-myc mice onto an lpr (CD95 receptor mutant) background,

the rates of tumor development and phenotypic properties, including levels of apoptosis, were observed to be indistinguishable from CD2-myc controls. In addition, tumor cell lines derived from mice expressing inducible Myc showed similar rates of inducible apoptosis regardless of their lpr genotype. These results are consistent with the conclusion that activation of cMyc and loss of Fas do not collaborate in T lymphoma development, and that c-Myc-induced apoptosis in Tcells occurs by Fas-independent pathways (Cameron et al., 2000). There is evidence to suggest that the CD95/Fas death receptor pathway is an integral part of the apoptotic response associated with the end of the normal terminal myeloid dierentiation program, and that deregulated c-myc expression can activate this signaling pathway prematurely (Amanullah et al., 2002). It appears, then, that in myeloid cells, the CD95/Fas apoptotic pathway might function in at least two ways to maintain homeostatic cell numbers. First, it contributes to the elimination of dierentiated myeloid cells at the end of their useful lifespan. And second, CD95/Fas may sensitize dierentiating cells to the presence of inappropriately expressed positive regulators of proliferation, such as c-Myc, and thereby facilitate the destruction of these cells. If unchecked, such aberrant cells could, by acquiring additional mutations, progress to a leukemic phenotype. Effect of concomitant constitutive expression of a positive regulator of myeloid differentiation and c-myc Since deregulated c-Myc both blocks dierentiation and its associated growth arrest, it can be asked if positive regulators of dierentiation can act as tumor suppressors when co-expressed with c-Myc. This type of study should yield new insights into the regulation of terminal dierentiation, progression to the tumorigenic state, and provide additional tools to design drugs for dierentiation therapy. Early growth response gene (egr) 1, a member of the egr family of genes that encode for zinc-®nger transcription factors, plays a role in the development, growth control and survival of several cell types, including T and B cells, neuronal cells, and myeloid cells (Beckmann and Wilce, 1997; Krishnaraju et al., 1995, 1998; McMahon and Monroe, 1996; Nguyen et al., 1993). In this laboratory it has been demonstrated that Egr-1 stimulates development of hematopoietic progenitor cells along the macrophage lineage at the expense of the granulocyte and erythroid lineages (Krishnaraju et al., 2001). Egr-1 was found to be a macrophage dierentiation primary response gene. Its ectopic expression in M1 cells activated the macrophage dierentiation program in the absence of dierentiation inducer; this included the appearance of morphologically dierentiated cells, decreased growth rate, and downregulation of c-myc mRNA. In addition, ectopic expression of egr-1 in M1 cells dramatically increased the sensitivity of the cells for IL-6 induced dierentiation, allowed a higher propor-


tion of M1 cells to become terminally dierentiated, and decreased M1 leukemogenicity in vivo (Krishnaraju et al., 1998). Ectopic expression of egr-1 abrogated the block in terminal dierentiation imparted by c-myc, resulting in cells that have the characteristics of functionally mature macrophages. However, the cells failed to adhere to the culture dish, did not become G0/G1 arrested, and continued to cycle for several days, prior to undergoing programmed cell death (Shafarenko, Liebermann and Homan, unpublished results). The ability of Egr-1 to promote terminal myeloid dierentiation appears to be dominant to c-Myc. Although c-Myc prevented growth arrest, the terminally dierentiated cells succumbed to apoptosis. Therefore, Egr-1 behaves like a tumor suppressor, at least within the context of this experimental system. In addition, these observations reveal that cycling cells can undergo myeloid dierentiation, and, therefore, that deregulated c-Myc does not interfere with dierentiation by virtue of preventing growth arrest. M1 cells ectopically expressing the BCR-ABL transgene also undergo macrophage dierentiation without arresting proliferation; interestingly, these cells continued to express high levels of c-myc mRNA (Cambier et al., 1999) Egr-1 and/or Egr-1 target genes may provide important tools for dierentiation therapy in some leukemias. Analysis of gene expression for the dierent established M1 variant cell lines should lead to an understanding of how Egr-1 overrides the c-Mycmediated block in myeloid dierentiation, which cMyc target genes participate in blocking dierentiation, and which promote proliferation. A combination of in vitro and in vivo moleculargenetic approaches have provided evidence for the many roles AP-1 (Fos/Jun) transcription factors play in the development of hematopoietic precursor cells along most, if not all, the hematopoietic cell lineages, including the monocyte/macrophage, granulocyte, megakaryocyte, mastocyte and erythroid lineages (Liebermann et al., 1998). Fos/Jun have been implicated as positive regulators of both apoptosis and terminal dierentiation in hematopoietic progenitor cells of the myeloid lineage (Liebermann et al., 1998; Lord et al., 1993). Activation of the foser transgene product partially abrogates the block imparted by deregulated c-myc on myeloid dierentiation, and also increases the sensitivity of the cells to respond to dierentiation factors (Shafarenko, Gregory, Liebermann and Homann, unpublished results). These ®ndings predict that genetic lesions which abolish cfos expression would cooperate with deregulated c-myc in leukemogenesis. Transcription regulators of c-myc expression Deregulated expression of c-myc prevents dierentiation of many cell types, can cause unscheduled apoptosis, can induce genomic instability (Felsher and Bishop, 1999a), and is associated with many tumors.

Therefore, precise regulation of c-myc expression is critical for maintaining normal hematopoiesis. Several dierent transcription factors have been implicated in the down-regulation of c-myc expression during dierentiation. The CCAAT/enhancer binding protein a (C/EBPa) is a basic leucine zipper transcription factor that is highly expressed in many dierentiated cell types, including granulocytes, hepatocytes and adipocytes, and it has been shown to be essential for granulocytic dierentiation (Zhang et al., 1997). c-Myc and C/EBPa have opposing eects in cells; c-Myc is expressed in proliferating cells and C/EBPa is expressed in dierentiated cells. It has also been shown that c-Myc represses C/EBPa (Li et al., 1994), and, recently, it has been demonstrated that C/EBPa negatively regulates cmyc transcription (Johansen et al., 2001). Not surprisingly, in certain myeloid leukemias in which C/EBPa expression has been observed to be low, c-myc expression is elevated (Johansen et al., 2001). Another transcription factor that regulates c-myc expression is the multifunctional multivalent zinc-®nger factor CTCF, which mediates repression or activation of many genes, and plays a role in growth suppression (Rasko et al., 2001). c-myc is among CTCFs many target genes, and is repressed by it. During dierentiation of human myeloid cells CTCF down-regulation is delayed and less pronounced than that of c-myc (Delgado et al., 1999). Consistent with the role of CTCF in growth suppression and inhibition of c-myc expression, there is evidence that loss of CTCF function is associated with cancer development (Feinberg, 2001; Ohlsson et al., 2001). B lymphocyte-induced maturation protein-1 (BLIMP-1) has been shown to be a key regulator of terminal dierentiation in myeloid cells and B lymphocytes. It is induced during myeloid dierentiation, and has been shown to repress c-myc expression in the human myeloid cell line U937 (Chang et al., 2000). In human promyelocytic leukemia HL-60 cells, induction of dierentiation along either the monocytic or granulocytic pathway blocks c-myc transcription. Protein kinase C plays a critical role in the dierentiation response of HL-60 cells to PMA, retinoic acid and 1,25-dihydroxyvitamin D3. Brie¯y activating PKC is sucient to block c-myc transcription, and the regulatory factor X (RFX) consensus binding site (X box) of c-myc intron 1 is essential for the downregulation of c-myc by PKC. PMA treatment of HL60 cells induced nuclear translocation of RFX1. RFX proteins, ubiquitously expressed in mammalian cells, can up- or down-regulate transcription of target genes (Chen et al., 2000). How negative transcriptional control of c-myc is regulated during dierentiation of normal blood cells, and the role of these dierent negative regulators in normal dierentiation is not clear, nor is it known what the consequences are following loss of activity of these regulators, especially with regard to oncogenic transformation of blood cells. In addition, positive regulators of c-myc transcription, such as c-Myb, have

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been shown to be involved in myeloid leukemogenesis (Schmidt et al., 2000). To maintain precise regulation of c-myc expression must require the interaction of a complex array of dierent trans regulators, both positive and negative. Altering subsets of regulators can have extensive rami®cations with regard to the proliferative and survival capabilities of the cells, altering hematological homeostasis, and participating in dierent disease states including anemias, leukemias and lymphomas. Myc and tumorigenesis Alterations in the level of c-myc expression or protein structure are associated with many malignancies in humans and animals (Askew et al., 1991; Evan and Littlewood, 1993). In many instances it is not clear if alterations in c-myc expression and/or function play a role in either initiation or progression of the disease in humans, in contrast to the clear cut case of Burkitt's lymphoma (BL) (Nesbit et al., 1999). Myc genes have been shown to be activated by a variety of genetic alterations including chromosomal translocation, proviral insertion, retroviral transduction and gene ampli®cation, although most often the basis for altered c-myc expression is not yet understood (Boxer and Dang, 2001; Ryan and Birnie, 1997). Non random somatic mutations frequently occur in BLderived c-myc genes (Bhatia et al., 1993, 1995; Nesbit et al., 1999; Rabbitts et al., 1983). Alterations in the protein can in¯uence association with p107, which mediates negative regulation of c-Myc transactivation, as well as protein stabilization, which can augment cMyc levels (Beijersbergen et al., 1994; Boxer and Dang, 2001; Gu et al., 1994; Nesbit et al., 1999). In addition, mutations in the noncoding region have been associated with loss of transcriptional attenuation and longer half life of transcripts (Boxer and Dang, 2001; Nesbit et al., 1999). Double minute chromosomes (DMs), cytogenetic hallmarks of ampli®ed genes, are infrequently observed in acute and chronic myeloid leukemia; c-myc is commonly found to be retained in the DMs (Crossen et al., 1999; Nesbit et al., 1999; O'Malley et al., 1999). Deregulated expression of c-myc is not sucient to induce transformation of primary cells, and the long held assumption that it is sucient for immortalization is disputed (Adams et al., 1999; Henriksson and Luscher, 1996; Ryan and Birnie, 1997). However, cmyc and ras have been shown to cooperate in both transformation and tumorigenesis in vivo (Henriksson and Luscher, 1996; Ryan and Birnie, 1997). Results obtained from studies employing mouse models are consistent with the need for at least one, and possibly two, mutations in addition to deregulated c-myc for malignant tumor formation (Adams et al., 1999; Nesbit et al., 1999). Using transgenic mice expressing an inducible c-myc transgene in their hematopoietic cells, Felsher and Bishop (1999b) showed that expression of c-myc led to

Oncogene

malignant lymphoid and myeloid tumors, and that subsequent inactivation of the transgene resulted in regression of some of the established tumors. This work demonstrated that although tumorigenesis is a multistep process, some tumors were dependent upon the initiating Myc lesion for malignancy. Apoptosis associated with inappropriate c-myc expression, as with other oncogenes such as E1A, limits the tumorigenic eect of the oncogene. Mechanisms which inhibit apoptosis should enhance or promote tumorigenesis. The observed oncogenic synergy between Myc and bcl-2 demonstrates that this is indeed the case. Bcl-2 has been shown to inhibit Mycmediated apoptosis in some but not all cell systems, while having no eect on the proliferative function of c-Myc (Bissonnette et al., 1992; Fanidi et al., 1992). Bcl-2 cooperated with c-Myc to promote proliferation of B-cell precursors, and some of these cells became tumorigenic. This cooperation was exhibited in vivo as well, where the doubly transgenic Eu-bcl-2/myc mice developed tumors much faster than Eu-myc mice (Strasser et al., 1990). Tumors arising in Eu-bcl-2/myc and Eu-myc mice are clonal and appear only after a latency period, indicative of the requirement for further somatic mutations for tumor formation. Other genes besides bcl-2 and ras cooperate with cmyc in tumorigenesis. In Eu-v-abl mice, c-myc and Nmyc were tagged as oncogenic partners (Haupt et al., 1993). In addition, myb, pim-1, bmi-1/bla-1, pal-1, and raf-1 have been shown to cooperate with c-myc (Davies et al., 1999; Evan and Littlewood, 1993; Packham and Cleveland, 1995), where some of these genes, namely, pim1, raf and abl, exhibit anti-apoptotic activity. The network of c-Myc interacting proteins is quite extensive, and its functions are most likely modulated by these dierent proteins. In addition, DNA binding by c-Myc requires heterodimerization with Max, and Max also interacts with a network of proteins, further adding to the complexity of gene regulation by c-Myc. Alterations in the expression and/or function of some of the c-Myc and Max interacting proteins appear to eect the neoplastic process. MM-1 has been shown to interact with the amino terminus of c-Myc, and to repress E-box dependent transcriptional activity of c-Myc (Mori et al., 1998). Recently, it has been reported that a speci®c point mutation in MM-1 occurs frequently in cells from lymphoma, leukemia and tongue cancer, and that this mutation (A157R) also abrogated the inhibitory functions of MM-1 with regard to c-Myc-mediated transcriptional activation. The wild-type MM-1, but not A157R, arrested the growth of 3Y1 cells. In addition, the human MM-1 gene has been shown to map at chromosome 12q12-12q13, a site where many chromosomal abnormalities in cancer cells have been reported. The authors suggest that MM-1 may function as a tumor suppressor by virtue of negatively regulating the transcriptional activity of c-Myc. (Fujioka et al., 2001) Mice with homozygous deletions of mxi1 are predisposed to skin tumors and lymphoma. The spleens of mxi17/7 mice were slightly enlarged


compared to age-matched controls. At about one year of age, some of the mxi17/7 mice developed malignant lymphomas of B cell origin, whereas none of the mxi1+/+ mice became ill. In addition, treatment with the carcinogen 9,10-dimethyl-1,2-benzanthracene (DMBA) resulted in higher frequencies of squamous cell carcinoma of the skin and malignant lymphoma in the mxi17/7 mice compared to wild type controls; mxi1 is expressed in these two cell types. Loss of function of Mxi1 also enhanced the incidence of tumors of INK4A7/7 mice, with lymphomas being one of the predominant tumor types observed (Schreiber-Agus et al., 1998). Although Mxi1 acts as a tumor suppressor in vivo, studies using mad17/7 mice provide no evidence that Mad1 is also a tumor suppressor (Foley et al., 1998). There are many more known, and probably also many unidenti®ed, c-Myc interacting proteins, which can either enhance or repress c-Myc activity, and it is likely that at least some of these will also participate in tumorigenesis. The apoptotic capability of oncogene expressing cells, including c-myc, accounts for the sensitivity of many primary tumors to anticancer agents relative to their normal counterparts. This is the basis for the success of many cancer therapies. However, further mutations often eliminate the apoptotic response, rendering the treatment ineective. Since many Mycexpressing cells require p53 to undergo apoptosis, the p53 status of tumor cells will determine if the cell responds to a particular therapeutic treatment. Similarly, activation of genes encoding for proteins such as Bcl-2, which inhibit the apoptotic response would also render cells resistant to certain treatments. (Homan and Liebermann, 1998) Myc target genes C-Myc in¯uences a variety of cellular functions, including cell cycle regulation, metabolism, apoptosis, dierentiation and cell adhesion, and deregulated expression participates in tumorigenesis. It is still not understood how c-Myc exerts its biological eects. There is ample evidence that c-Myc binds E boxes and transactivates genes. In addition, c-Myc can repress transcription. How c-Myc regulates its many target genes is not clear. Since c-Myc is part of a complex network of interacting proteins, and dierent regions of c-Myc appear to be important for dierent biological functions, it is probable that c-Myc regulates expression of target genes by several dierent mechanisms. In spite of the fact that many laboratories have identi®ed c-Myc target genes, employing dierent strategies and often using dierent cell types, there is

still no consensus about which target genes are responsible for which Myc-mediated functions (Boxer and Dang, 2001; Dang, 1999). It is dicult to establish if a putative target gene is physiologically relevant for a speci®c biological function, since expression of the gene by itself may not be sucient to elicit the response. It is likely that subsets of target genes cooperate to elicit a speci®c response to Myc. To ascertain if expression of a particular target gene is essential for a Mycmediated phenotype, blocking expression, by either antisense methodology or dominant negative alleles, of a given target gene can be done. Among the dierent strategies employed to identify target genes, are dierential expression and the presence of either an E box or initiator element in promoter regions. Comprehensive reviews of the dierent strategies, types of genes identi®ed, as well as a discussion of direct and indirect target genes are available (Boxer and Dang, 2001; Dang, 1999). The use of micro arrays, appropriate cell systems, and determination of physiological relevance will lead to the identi®cation and assignment of target genes to the dierent physiological roles played by c-Myc, including both normal functions as well as the initiation and progression of dierent neoplasias.

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Concluding remarks C-Myc in¯uences multiple biological activities, some in the context of appropriate expression and some detected by deregulated expression. These include cell cycle regulation, metabolism, apoptosis, dierentiation, cell adhesion, and tumorigenesis. Tightly regulated cmyc expression is crucial for normal hematopoiesis, and alterations in the level of c-myc expression or protein structure are associated with many hematological malignancies. Myc function can also be in¯uenced by its network of interacting proteins. Most probably, dierent biological responses are elicited by dierent overlapping subsets of target genes, both induced and suppressed, and c-Myc regulates expression of target genes by several dierent mechanisms. What ultimately determines the cell's response to c-Myc is the speci®c biological setting, including the expression of other oncogenes and tumor suppressor genes. Understanding how c-Myc controls cellular phenotypes, including the leukemic phenotype, will provide novel tools for designing drugs to promote dierentiation and/or apoptosis of leukemic cells. Acknowledgments This research was supported by National Institute of Health Grant RO1 CA81168 (to B Homan).

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