c-MYC is the prototype for oncogene activation by chromosomal translocation. In contrast to the tightly regulated expression of c-myc in normal cells, c-myc is frequently deregulated in human cancers. Herein, aspects of c-myc gene activation and the function of the c-Myc protein are reviewed. The c-myc gene produces an oncogenic transcription factor that affects diverse cellular processes involved in cell growth, cell proliferation, apoptosis and cellular metabolism. Complete removal of c-myc results in slowed cell growth and proliferation, suggesting that while c-myc is not required for cell proliferation, it acts as an integrator and accelerator of cellular metabolism and proliferation.
Regulation of c-myc expression
The c-myc gene is regulated by complex mechanisms at both the transcriptional and post-transcriptional levels (Bornkamm et al., 1988; Spencer and Groudine, 1991). Four transcriptional promoters have been identified, but RNA initiated at the P2 promoter usually contributes to 80–90% of total c-myc steady-state RNA in normal cells (Taub et al., 1984). The c-myc gene comprises three exons. Exon 1 contains two promoters and is noncoding. Exons 2 and 3 encode the Myc protein with translation initiation at nucleotide 16 of exon 2.
The regulation of c-myc expression has been the subject of many studies. Numerous transcription factors have been reported to interact with regions of the c-myc gene, but results of different studies have yielded conflicting data (Marcu et al., 1992; Spencer and Groudine, 1991). This is due at least in part to the fact that different cell types and promoter constructs have been used. Several DNase I hypersensitive sites have been identified 5′ and internal to the c-myc gene (Bentley and Groudine, 1986; Dyson et al., 1985; Dyson and Rabbitts, 1985). We have shown that transcription factors bind in vivo to a number of these sites in the c-myc promoter (Arcinas and Boxer, 1994). NF-κB is an important regulatory factor for both the murine (Kessler et al., 1992; La Rosa et al., 1994) and human (Ji et al., 1994) c-myc promoters in B cells. The c-myc gene is a target of the APC/β-catenin/Tcf pathway in colon carcinoma cells (He et al., 1998), but the significance of this regulatory pathway in lymphocytes is not clear. The role of APC and catenins in colon carcinogenesis will be discussed below.
In addition to transcription initiation, transcription elongation plays an important role in the regulation of c-myc expression. The down-regulation of c-myc mRNA during differentiation of different cell types occurs by a block to transcription elongation (Bentley and Groudine, 1986; Eick and Bornkamm, 1986; Nepveu and Marcu, 1986). Kinetic nuclear run-on experiments and mapping of single stranded DNA in vivo have revealed that the polymerase II complex is paused at +30 relative to the major transcription initiation site (Krumm et al., 1992; Strobl and Eick, 1992).
Translocations of c-myc
The c-myc gene is translocated to one of the immunoglobulin loci in virtually all Burkitt's lymphomas. The typical translocation of c-myc into the immunoglobulin heavy chain locus is observed in about 80% of Burkitt's lymphomas. The variant translocations of c-myc into either the κ or λ light chain locus each occur at a frequency of about 10%. Burkitt's lymphomas are divided into endemic (African-derived) and sporadic. The vast majority of endemic Burkitt's cases are associated with Epstein–Barr virus (EBV) infection, and while it is likely that EBV plays a causal role, the mechanisms involved have not been completely defined. As discussed below, the breakpoints are often different in these two groups of lymphomas. Burkitt's lymphoma accounts for approximately 30% of all HIV-associated lymphomas. Translocations of c-myc into one of the immunoglobulin gene loci are also observed in diffuse large cell lymphoma. Some of these lymphomas also show a t(14;18) translocation of the bcl-2 gene into the immunoglobulin heavy chain gene and may represent lymphomas that have transformed from low-grade follicular lymphoma.
The c-myc gene is translocated into one of the T cell receptor loci in some cases of T-cell acute leukemia. More recently, translocations of c-myc into one of the immunoglobulin genes have been found in multiple myeloma (Drach et al., 2000; Shou et al., 2000). These abnormalities include complex translocations and insertions and are unlikely to be caused by errors in VDJ or class switch recombination or somatic hypermutation (Shou et al., 2000). While the translocation of c-myc into the immunoglobulin or T cell receptor locus is believed to be one of the primary events in malignant transformation in Burkitt's lymphoma and in T-ALL, it is most likely a secondary event associated with progression in multiple myeloma. A summary of the diseases frequently associated with c-myc translocations is shown in Table 1.
Translocation breakpoints in Burkitt's lymphoma
Translocation breakpoints in Burkitt's lymphomas fall into three classes defined by their location with respect to c-myc (Cory, 1986): within the c-myc gene (class I), immediately 5′ to the c-myc gene (class II), and distant (class III). Class III breakpoints can be more than 100 kb from the c-myc gene. Thus, in order to detect the translocations by physical linkage to the immunoglobulin locus, pulsed field rather than conventional electrophoresis must be used.
The class I breakpoints cluster within exon 1 and the first intron. These translocations may disrupt regulatory signals for c-myc. Many of the class II breakpoints are within a few kilobases 5′ to exon I. Several protein binding sites have been detected in the 5′ flanking region by in vitro assays and have been proposed to be positive or negative regulators of transcription.
In endemic Burkitt's lymphoma, the translocation breakpoint usually occurs 5′ of the c-myc gene, and the distance can be quite large. In these cases, the c-myc gene is intact. In sporadic Burkitt's lymphoma, the breakpoint is often in the first exon or the first intron (Magrath, 1990; Shiramizu et al., 1991). The c-myc coding region consisting of the second and third exons is always intact, however. It is likely that the pathogenesis of endemic and sporadic Burkitt's lymphoma differ, and the state of differentiation of the target cell for transformation is most likely different in the two types of lymphoma.
The breakpoint on the immunoglobulin gene usually occurs at the VDJ region in endemic Burkitt's lymphoma while a breakpoint at the class switch region is most common in sporadic Burkitt's lymphoma (Magrath, 1990; Shiramizu et al., 1991). In HIV associated Burkitt's lymphomas, the translocation breakpoint also occurs at the class switch region. Examples of some translocation breakpoints are shown in Figure 1. The intron enhancer in the immunoglobulin heavy chain gene is located on the same chromosome as the c-myc gene in endemic Burkitt's lymphoma, but it is located on the reciprocal translocation product in sporadic Burkitt's lymphoma and thus is not available to deregulate c-myc expression. As discussed in the next section, there are several enhancers located 3′ of the immunoglobulin heavy chain gene, which are always located on the same chromosome as the translocated c-myc gene.
Deregulation of c-myc expression in Burkitt's lymphoma
Translocation of the c-myc gene to the heavy or light chain immunoglobulin locus is seen in virtually every case of Burkitt's lymphoma and in many cases of transformed lymphomas. Transcription of the translocated c-myc is greater than that seen in resting B cells; however, in some cases the magnitude of the increase is no more than that observed in actively dividing but non-malignant B cells, such as those infected with the EBV. The normal c-myc allele is usually transcriptionally silent in Burkitt's lymphomas (ar-Rushdi et al., 1983; Cory, 1986; Hayday et al., 1984; Nishikura et al., 1983), and thus the only Myc protein in most Burkitt's cells is derived from the translocated c-myc allele. In Burkitt's cell lines, transcription of the translocated c-myc is preferentially initiated further upstream at promoter P1 instead of at P2 (Strobl and Eick, 1992; Strobl et al., 1993; Taub et al., 1984). The cause of this promoter shift is not known, but the immunoglobulin heavy chain gene enhancers are required for induction of the promoter shift. All of these changes have been interpreted as evidence for the activation or deregulation of the translocated c-myc gene (Bernard et al., 1983; Erikson et al., 1983; Leder et al., 1983; Taub et al., 1984).
It does not appear that the interruption of c-myc regulatory sequences by class I and II breakpoints is sufficient for malignant transformation. Class III breakpoints are distant from the c-myc gene and do not affect the 5′ flanking sequence. It is likely that regulatory elements from the immunoglobulin locus are responsible for the deregulation of c-myc expression. It is possible that the immunoglobulin elements require certain c-myc regulators for the interaction to occur. Depending on the class of translocation, different c-myc elements may be used. For instance, in the Ramos cell line, the 5′ flanking sequence is located on a different chromosome from the c-myc gene, and we have observed in vivo occupancy of an NF-κB site in the c-myc first exon on the translocated allele. In the Raji cell line this region of the first exon is deleted from the translocated c-myc gene, and we found in vivo occupancy of an NF-κB site in the 5′ flanking region of c-myc (Ji et al., 1994). We have also found an in vivo footprint over a potential Nm23 binding site in the c-myc promoter (Ji et al., 1995). Two sites in the P1 promoter region that are required for the κ immunoglobulin gene activation of the P1 promoter have been identified. In transfection studies the TATA box and an Sp1 site are both essential and sufficient for this activation (Geltinger et al., 1996).
The deregulation of c-myc gene expression is thought to play a causal role in malignant transformation. Transgenic mice, which carried c-myc linked to the immunoglobulin heavy chain gene intron enhancer, developed clonal B cell malignancies (Adams et al., 1985; Nussenzweig et al., 1988). Furthermore, when lymphoblastoid cells (immortalized by EBV) were transfected with a constitutively expressed c-myc gene, the cells became tumorigenic in nude mice (Lombardi et al., 1987).
Mechanisms of deregulation of c-myc in Burkitt's lymphoma
It is likely that the translocated c-myc gene falls under the influence of immunoglobulin gene regulatory elements. Although transgenic mice with the c-myc gene linked to the immunoglobulin heavy chain intron enhancer develop B cell malignancies, these studies shed no light on the biological role of the intron enhancer in the t(8;14) translocation. They do demonstrate that high level expression of c-myc in B cells by a regulatory element, which is active in B cells, can lead to development of a B cell malignancy. In fact, the malignancy that develops in these mice does not resemble Burkitt's lymphoma, particularly because the malignant cells in these mice are at the pre-B cell stage of differentiation, as opposed to the mature B cells in Burkitt's lymphoma. These mice served to demonstrate that c-myc was an oncogene in B cells, but they do not provide a useful model for Burkitt's lymphoma. Mice carrying a yeast artificial chromosome (YAC) containing the c-myc gene linked to a portion of the immunoglobulin heavy chain gene locus also developed B cell malignancies (Butzler et al., 1997). However, only the intron enhancer was present, and the 3′ enhancers were not included in this model. In fact, the intron enhancer was deleted, and the mice still developed B cell malignancies (Palomo et al., 1999). It is likely that the YAC has integrated into a transcriptionally active region of the chromosome, and additional regulatory elements are not required for expression of c-myc.
In a number of Burkitt's lymphomas, the immunoglobulin intron enhancer is not available to the c-myc gene because it is located on the other chromosome in the translocation. The best candidate for deregulation of the c-myc gene is the 3′ enhancer, which has been found in the kappa and heavy chain immunoglobulin loci (Judde and Max, 1992; Muller et al., 1990). Matrix association regions (MARs) are located near the immunoglobulin intron enhancer in the murine and human kappa loci (Whitehurst et al., 1992) and in the murine heavy chain locus (Cockerill et al., 1987). MARs are frequently located near enhancers and some appear to have enhancer activity. They often contain topoisomerase II sites and may therefore be involved in both transcriptional enhancement and swiveling at the base of chromosome loops.
Several enhancers have been shown to be important for expression of the immunoglobulin heavy chain gene. Four B-cell-specific and cell-stage-dependent DNase I hypersensitive sites, HS1 to HS4, are located 10–35 kb 3′ of the Cα gene (Madisen and Groudine, 1994; Michaelson et al., 1995; Pettersson et al., 1990; Saleque et al., 1997). These regions have been shown to function as a locus control region in B cells (Madisen and Groudine, 1994). Enhancers have been located downstream of two human Cα genes, and these regions share some homology with the murine HS1-4, but only limited functional studies have been performed on the human enhancers. The 3′ region of the immunoglobulin heavy chain locus is linked to the translocated c-myc gene in all t(8;14) translocations in Burkitt's lymphoma, and it is likely that this region plays a role in the deregulated expression of the translocated c-myc gene.
In addition to a shift in promoter usage from P2 to P1, the block to transcription elongation is absent on the translocated c-myc gene. Nuclear run-on studies have shown that the initiation rate at the c-myc P1 promoter is not increased but that the number of paused polymerase complexes near the P2 promoter is markedly decreased (Strobl et al., 1993). Constructs consisting of the c-myc gene linked to enhancer regions from the different immunoglobulin genes have been used to study the deregulation of c-myc transcription. Linkage of c-myc to the MAR, intron enhancer, constant κ gene and the 3′ enhancer resulted in increased c-myc expression and a shift in promoter usage from P2 to P1 (Polack et al., 1993). Linkage to either the intron or the 3′ enhancer alone resulted in less activation of c-myc, and there was no promoter shift. All t(2;8) translocations between c-myc and the κ light chain gene result in colocalization of the intron and 3′ enhancers with c-myc on the translocated chromosome. As discussed above, the intron enhancer on the immunoglobulin heavy chain locus is usually located on a different chromosome from the translocated c-myc gene. Linkage of c-myc to the 3′ enhancers (HS1234) from the immunoglobulin heavy chain region resulted in high level expression of c-myc, the promoter shift, and loss of the block to transcription elongation at the P2 promoter (Madisen and Groudine, 1994). The intron enhancer was not required for these effects. Similar results have been obtained with enhancer regions from the λ light chain gene (Gerbitz et al., 1999). More recently, it has been shown that the 3′ enhancers from the immunoglobulin heavy chain locus increase expression from the c-myc P2 promoter by an increase in histone acetylation. However, this increase in acetylation does not explain the HS1–4 activation of transcription from the P1 promoter (Madisen et al., 1998).
Investigations into the specific sites in the immunoglobulin gene enhancers that are required for the deregulation of c-myc expression have been performed. Our studies showed that an NF-κB site in HS4 from the immunoglobulin heavy chain locus was required for the enhancer activity of this region when it was linked to the c-myc promoter. The NF-κB site was also required for the shift in transcription initiation from P2 to P1. The promoter shift due to HS4 was lost when the NF-κB site was mutated, although it is clear that regions of HS1, 2 and 3 also contributed to the full promoter shift (Kanda et al., 2000). Most of these studies have been performed with the murine immunoglobulin heavy chain gene. We identified an NF-κB site in the human HS4 enhancer region and showed that it was active with the c-myc promoter (Kanda et al., 2000). An NF-κB site in the κ light chain gene intron enhancer was shown to be important for the deregulation of c-myc expression in the t(2;8) translocation. In addition, a binding site for PU.1/Spi-1 in the κ 3′ enhancer was also required. Mutation of both the NF-κB and PU sites resulted in the loss of activation of the c-myc P1 promoter (Wittekindt et al., 2000).
These findings support a model in which sequences present in the immunoglobulin gene locus deregulate expression from the cis-linked c-myc allele by promoting interactions between c-myc and immunoglobulin gene regulatory elements that affect c-myc initiation and elongation. As proposed in Figure 2, interactions between multiprotein complexes assembled on both the c-myc promoter and immunoglobulin enhancers may occur with looping out of the intervening DNA sequence. It should be noted, however, that the translocation breakpoint in many sporadic Burkitt's lymphomas separates the c-myc promoter from the coding region (Pelicci et al., 1986; Shiramizu et al., 1991). In these cases, the regulatory elements of the immunoglobulin gene enhancers apparently activate c-myc transcription without interaction with the c-myc promoter elements. Transcription often initiates in the first intron of c-myc in these sporadic Burkitt's lymphomas.
Sequence mutations in the translocated c-myc gene
Most translocated c-myc alleles from Burkitt's lymphoma cell lines contain sequence alterations, often point mutations or deletions (Cesarman et al., 1987; Rabbits et al., 1984; Siebenlist et al., 1984; Taub et al., 1984; Yu et al., 1993). The translocated allele is located in the hypermutable immunoglobulin locus and thus may be subject to somatic mutations at an increased frequency (Bemark and Neuberger, 2000). Many mutations are located near the two promoters in exon I or near the exon I–intron I boundary. These regions are important regulatory sites, which demonstrate protein binding in vivo. Mutations in the exon I/intron I boundary region of the translocated c-myc gene have been found in diffuse large cell lymphomas with either the t(8;14) or t(8;22) translocations (Bradley et al., 1993). Although mutations are found in potential c-myc regulatory regions in many cases of Burkitt's lymphoma, there is no evidence that they are the critical events in the activation of c-myc or even that the same site is mutated in the majority of lymphomas.
Hotspot mutations and activation of c-myc in human neoplasias
In addition to chromosomal translocations that activate c-myc in lymphoid neoplasms and regulatory sequence mutatations, the coding sequence also bears hotspot non-conserved mutations (Figure 3) (Bhatia et al., 1993; Yano et al., 1993). The c-Myc hotspot mutations are most likely the result of somatic hypermutation, a hallmark of B-cell maturation in the germinal center. The consequences of the c-Myc mutations are now better appreciated. There is evidence supporting the inhibition of c-Myc function by the pocket protein p107 (Beijersbergen et al., 1994; Gu et al., 1994; Hoang et al., 1995). Two studies suggest that mutations in the N-terminal region of c-Myc result in the inability of p107 to inhibit c-Myc function, although this concept remains controversial.
One of the c-Myc Box I hotspot is Thr-58, which may be phosphorylated or O-linked glycosylated (Chou et al., 1995; Lutterbach and Hann, 1994). Phosphorylation of Thr-58 appears to be required for proteosome-mediated degradation of c-Myc, such that mutation of Thr-58 to Ala results in protein stabilization (Bahram et al., 2000; Flinn et al., 1998; Gregory and Hann, 2000). Furthermore, activated RAS, which cooperates with c-Myc in transformation, inhibits the phosphorylation of this region and causes c-Myc protein stabilization (Sears et al., 1999). In several studies, mutations at or around Thr-58 appear to augment c-Myc mediated transformation. c-Myc mediated apoptosis does not generally appear to be affected or may be diminished (Chang et al., 2000; Conzen et al., 2000; Hoang et al., 1995). While the role of O-linked glycosylation of c-Myc remains to be established, it appears that hotspot mutations at or around Thr-58 result in protein stabilization that may further augment c-Myc levels and presumably Myc-mediated transcriptional regulation.
In addition to activation of the c-myc gene in lymphoid tumors through chromosomal translocation and point mutations, c-myc is frequently deregulated in a variety of human tumors (Nesbit et al., 1999). C-myc gene amplication accounts for a portion of human breast, lung, ovarian and prostate carcinoma as well as medulloblastoma. C-myc expression is more frequently deregulated, however, in a large fraction of colon, gynecological tumors and melanoma.
The mechanism underlying deregulated c-myc expression in human cancers has not been well-characterized; however, the Wnt-Adenomatous Polyposis Coli (APC) pathway has been implicated in colon carcinogenesis. In particular, one study suggests that loss of APC, which mediates the proteosomal degradation of β-catenin, could result in an increase in β-catenin that can in turn activate the c-myc gene in cooperation with the transcription factor TCF4/LEF (He et al., 1998). While this pathway appears attractive as an explanation for augmented c-myc expression in colon cancer, a more recent study indicates that elevated c-Myc expression is unnecessary for β-catenin mediated cell transformation (Kolligs et al., 1999). In addition, γ-catenin (plakoglobin) appears to be more consistently connected with increased c-myc expression and cellular transformation (Kolligs et al., 2000). Despite the identification of c-myc as a downstream target of TCF4/LEF, β-catenin is not as robust as plakoglobin in activating c-myc gene expression. Hence, both plakoglobin and β-catenin may be downstream of the APC pathway and are both required for deregulated c-myc expression in colon carcinogenesis.
Functional domains of c-myc and c-myc transcriptional properties
The c-Myc protein belongs to the larger family of helix–loop–helix leucine zipper (HLHzip) transcription factors and is also a member of the Myc family of proteins (B-Myc, L-Myc and N-Myc) that are characterized by two conserved N-terminal regions, termed Myc Box I and Box II (Grandori et al., 2000; Henriksson and Luscher, 1996). The c-myc gene encodes several polypeptides. A 62 kDa protein is produced from a canonical AUG translational start site and a 64 kDa protein from an upstream CUG (Hann et al., 1992). Internal translational initiation results in a shortened 45 kDa form, termed MycS (Spotts et al., 1997). All three polypeptides heterodimerize with Max, an HLHzip protein, to bind target DNA sequences called E-boxes (Prendergast and Ziff, 1992). Together with Max, Myc regulates the transcription of target genes and induces cell growth, proliferation, or apoptosis and to inhibit cellular differentiation.
The N-terminal 143 amino acids and the C-terminal 140 amino acids of c-Myc are required for neoplastic transformation and inhibition of cellular differentiation (Dang, 1999). These two regions correspond to the N-terminal transactivation domain and the C-terminal DNA-binding and HLHzip dimerization domain. The c-Myc transactivation domain was identified through studies with GAL4 fusion proteins (Kato et al., 1990). The first 143 amino acids, which comprises of Myc Box I (residues 45–63) and Box II (residues 128–143), are sufficient for gene activation. Deletions of Box II do not affect transcriptional activation in transient reporter assays; however, Myc-mediated suppression of initiator-driven transcription is abrogated (Lee et al., 1996; Li et al., 1994). Hence, it has been proposed that Box II participates in transcriptional repression that appears to be required for neoplastic transformation. The function of Box II remains unclear, in particular since a recent study demonstrates that mutation of Box II resulted not only in loss of transcriptional repression but also in the inability to increase the expression of endogenous ornithine decarboxylase (ODC), a Myc target gene (Conzen et al., 2000). It is very likely that transient reporter assays do not fully reflect the transcriptional activities of c-Myc on genomic targets. Nevertheless, it stands to reason that Box II may mediate transcriptional repression and activates a specific subset of genes such as ODC.
The binding of the cofactor TRRAP, which associates with histone acetyl transferase activity, to Myc Box II region further suggests a role for Box II in transcriptional activation (McMahon et al., 1998, 2000). However, it is notable that mutations in Box II only result in the loss of activation of some but not all Myc target genes. As such, it is tempting to speculate that c-Myc might regulate different subsets of genes through different Myc regions and by different mechanisms. Perhaps Box II region is required for the activation of gene expression through histone acetylation, whereas Box II-independent transcriptional activation primarily depends on the recruitment of the transcriptional complex to genomic regions where histones are already hyper-acetylated. The use of specific c-Myc mutants in combination with arrayed gene expression analysis will hopefully provide insight into the mystery of Myc Box II.
The C-terminal region of c-Myc, which is required for transformation, contains a unique basic region that represents a distinct class of nuclear targeting sequences (Dang and Lee, 1988; Saphire et al., 1998). A second basic region, which weakly localizes proteins to the nucleus, turns out to be the DNA binding region of c-Myc that has a characteristic signature for binding the canonical 5′CACGTG-3′ central dinucleotide CG (Dang et al., 1992). A conserved Arg residue that directly contacts the central G is the signature of a subclass of Myc-like HLHzip proteins (Ferre-D'Amare et al., 1993). The HLHzip region represents a dimerization motif that allows for the formation of crisscrossing coiled-coil structures between c-Myc and its partner Max in a fashion that aligns the basic regions into the major groove of target DNA sequences. The loop region has been implicated in the contact of the protein to sequences flanking the core 5′CACGTG-3′ hexamer. Crystal structures of the HLHzip transcriptional factor USF suggests that certain HLHzip proteins could form higher order tetrameric complexes through the formation of a four-helical bundle via the leucine zippers (Ferre-D'Amare et al., 1994). It is possible that such complexes could account for the cooperative activity of c-Myc/Max on promoters that contain tandem E-boxes, although direct evidence for the role of tetramers in transcriptional regulation is lacking.
While most studies explore c-Myc transcriptional regulation in isolation, further understanding on how c-Myc cooperates with other transcriptional factors will be required to appreciate c-Myc's full activity in vivo. For example, the AP2 transcriptional factor negatively affects c-Myc (Gaubatz et al., 1995), whereas one report suggests that Sp1 cooperates with c-Myc (Kyo et al., 2000). C-Myc has also been implicated in altering C/EBP function through protein–protein interactions and not through directly contacting DNA binding sites flanking those of C/EBP (Mink et al., 1996). Hence, c-Myc function is likely to be significantly affected by nearby regulatory sequences as well as by protein–protein contacts that may be cell and tissue type specific. In addition, c-Myc transactivates other transcriptional regulators such as Coup, DP1, Fra1, HMG(Y)I, and Id2 (Coller et al., 2000; Lasorella et al., 2000; O'Hagan et al., 2000b; Schuhmacher et al., 2001; Wood et al., 2000). In this manner, c-Myc itself may hypothetically regulate promoters that also requires transcriptional factors produced from Myc targets, such as HMG(Y). In this case, an indirect Myc target gene could well be a direct target gene whose promoter requires both Myc and another transcription factor that is induced by Myc. The precedence for this has been reported for the expression of the myeloperoxidase gene that requires the transcription factors C/EBP and PU.1, whose expression is induced by C/EBP (Wang et al., 1999). With the emergence of DNA microarray technology to study c-Myc function, these lessons should be kept in mind if we aim to understand the full spectrum of Myc function in a variety of different tissues.
Max and Max-associated proteins
While truncated c-Myc, removed of its transcriptional regulatory domain, is capable of weakly homodimerizing in vitro and binding DNA, the full-length c-Myc polypeptide exists in a conformation that lacks DNA binding activity (Kato et al., 1992). DNA binding by c-Myc requires heterodimerization with Max, converting c-Myc from an inactive form to a heterodimer that is capable of binding E-boxes (Blackwood and Eisenman, 1991). Max, in contrast to c-Myc however, does not contain transregulatory regions, suggesting that its role is to switch on c-Myc's DNA binding capability. With this ability, it stands to reason that Max might be at the center of a complex network of protein–protein interactions where Max could switch on other HLHzip proteins. Indeed, Max is able to homodimerize as well as heterodimerize with Mad family proteins, Mga and Mnt (Grandori et al., 2000). Hence, the Max network of proteins further adds to the complexity of gene regulation by c-Myc.
In contrast to c-Myc, the Mad proteins contain the SID (Sin3-interacting domain) motif that is capable of recruiting Sin3A transrepression complexes and histone deacetylase activity (Ayer et al., 1993, 1996). The Mad/Max complexes, which bind E-boxes, could therefore antagonize Myc function by competing for target sites and render the associated genes silent through histone deacetylation. While it is attractive to hypothesize that Myc and Mad represents the yin and yang of the Max network, in which Myc activates the same genes that Mad silences, recent studies suggest that Mad/Max complexes may regulate a subset of genes that are not shared with c-Myc/Max (O'Hagan et al., 2000b). Furthermore, Mad family members appear to display differentiation specific expression such that only certain Mad proteins are expressed at specific stages of cellular differentiation (Queva et al., 1998).
The fact that Mad could antagonize Myc transcriptional activity suggests that it may inhibit Myc transforming activity (Ayer et al., 1993; Roussel et al., 1996). Indeed, in several model systems, Mad inhibits Myc-mediated neoplastic transformation (Koskinen et al., 1995; Lahoz et al., 1994; Schreiber-Agus et al., 1995). This further suggests the possibility that certain MAD genes might behave as tumor suppressor genes, the loss of which would allow Myc to function in an un-antagonized manner. Among the different Mad family members, only Mxi-1 (Mad2) to date has been implicated as a potential tumor suppressor. Homozygous deletion of Mxi-1 predisposes the knock-out mice to skin tumors and lymphoma (Schreiber-Agus et al., 1998). In addition, the human chromosomal region 10q24 bearing the Mxi-1 gene frequently display loss of heterozygosity in different human cancers including prostate (Prochownik et al., 1998) and the brain tumor, glioblastoma multiforme (Wechsler et al., 1997). However, the vast majority of the remaining allele in these tumors is not mutated. As such, if Mxi-1 functions as a suppressor, it must contribute to tumorigenesis through haploinsufficiency. It is also possible, although as yet not established, that the remaining Mxi-1 allele may be silenced through methylation.
Cellular functions and target genes of Myc
Despite the complexity of Max network of proteins, a common theme of tumorigenesis is deregulated c-Myc activity. Thus, to understand c-Myc physiologic and tumorigenic activity, we need to understand the gene expression programs that are induced by physiologically regulated c-Myc activity versus genes that are regulated by ectopic c-Myc expression. To understand these nuances, experimentally tractable models are required. Unfortunately, the study of c-Myc target genes has not been standardized as to the cell types used or the expression system employed (Cole and McMahon, 1999; Dang, 1999). Perhaps this is reflected by the fact that the vast majority of c-Myc responsive genes do not overlap between different studies. Whether these differences are due to different technical approaches or whether they reflect cell type differences remains to be established. Despite these shortcomings, a role for c-Myc in cellular functions has emerged (Figure 4). For the following discussion, specific Myc target genes verified by independent studies from several laboratories are used as examples.
Cell growth and cellular metabolism
C-Myc has been implicated in the transition of cells from the resting phase of Go (those deprived of growth factors in the experimental setting) into G1 and the traversal of cells through G1, in which cells accumulate mass in preparation for DNA synthesis (Roussel, 1998). Studies of hypofunctioning Myc in the Drosophila provide a slightly different understanding of c-Myc in cell growth and proliferation (Gallant et al., 1996; Schreiber-Agus et al., 1997). Hypo-functioning of Drosophila Myc (dmyc) results in small body size, a phenotype collectively termed Minutes. Intriguingly, most molecularly characterized Minutes are defective in specific ribosomal protein genes. In addition, it has been demonstrated that dmyc hypomorphs and Minutes, in general, are small not because there are fewer cells, but that the cells are smaller in size (Johnston et al., 1999). This observation suggests a role for c-Myc in cell mass accumulation and metabolism.
In mammalian systems, overexpression of c-Myc results in larger lymphocytes in vivo as well as in vitro (Iritani and Eisenman, 1999; Schuhmacher et al., 1999). In particular, induction of c-Myc expression in a human lymphocyte cell line under growth factor deprivation resulted in an increase in cell mass without cell proliferation (Schuhmacher et al., 1999). Likewise, Myc-induced cell mass accumulation has been demonstrated in primary murine fibroblasts (Beier et al., 2000). This induction is separable from Myc induction of E2F2 and cell cycle progression through the use of p27 null fibroblasts. Without affecting cyclin E-cdk2 activity, c-Myc induces E2F, S-phase entry and accumulation of cell mass in p27 null fibroblasts. In contrast, E2F2 was able to trigger S-phase entry without cell mass accumulation. An adenoviral gene delivery model of in vivo c-Myc expression also demonstrates the ability of c-Myc, but not E2F1, to cause liver cell hypertrophy without proliferation (Kim et al., 2000). In this case, within 4 days of intravenous delivery of Myc-expressing viruses, the murine livers were grossly enlarged with exuberantly large hepatocytes that express high levels of ribosomal protein genes. These studies all support the notion that c-Myc regulates the accumulation of cell mass as cells traverse from early to late G1 (Figure 5).
A number of c-Myc responsive genes appear to be involved in protein synthesis and hence might contribute to the cell growth phenotype (Schmidt, 1999). Since ribosomes account for a significant fraction of cell mass, regulation of their assembly is likely to affect cell mass accumulation. Myc responsive genes involved in ribosome biogenesis include nucleolin, nucleophosmin, BN51, fibrillarin, RNA helicase DDX18, and a variety of ribosomal protein genes (Coller et al., 2000; Greasley et al., 2000; Guo et al., 2000; Schuhmacher et al., 2001). Whether all of these genes are directly regulated by c-Myc remains uncertain, although in the case of nucleolin sufficient evidence have accumulated to support its direct regulation by c-Myc. In fact, nucleolin has appeared as a Myc target gene in several independent studies of different systems. What also remains uncertain is whether c-Myc affects rate-limiting steps of ribosomal assembly and whether c-Myc affects ribosomal RNA availability. One study suggests that c-Myc augments rRNA level post-transcriptionally (Gibson et al., 1992).
Several genes involved in translation have also been implicated as Myc target genes. In particular the translational initiation factors eIF2α, eIF3β, eIF4E, eIF4G, eIF5A, the translational elongation factor EF-2, amino acid transporter ECA39, as well as the aminoacyl-tRNA synthetases have appeared as Myc responsive genes (Coller et al., 2000; Guo et al., 2000; Rosenwald et al., 1993; Schuhmacher et al., 2001; Schuldiner et al., 1996). Taken together, different studies have implicated the protein synthetic machinery as a target for c-Myc regulation. Which of the rate-limiting steps for protein synthesis are affected by c-Myc remains unknown. Hence, additional studies are necessary to delineate the causal connection between c-Myc, its target genes involved in protein synthesis and the phenotype of cell mass accumulation.
For cells to accumulate mass from early to late G1, their biosynthetic capability must be coupled with appropriate energy metabolism. In several models of cell proliferation, it has been demonstrated that there is a significant increase in glycolytic metabolism before cells enter S phase. It is hypothesized that glycolytic metabolism, which has diminished production of reactive oxygen species as compared to oxidative phosphorylation, would protect DNA from oxidative damage (Dang and Semenza, 1999). Intriguingly, many cancers are also known to have a high rate of glucose uptake and lactate production, a phenomenon reported over seven decades ago and known as the Warburg effect. Also notable is that human tumors tend to have increased lactate dehydrogenase (LDH) A, and the serum LDH level has remained a major independent predictor of poor outcome for diverse types of human cancers. C-Myc not only direct regulates LDHA, but it also directly activates the expression of the glucose transporter 1 (GLUT1), phosphofructokinase, and enolase A (Osthus et al., 2000; Schuhmacher et al., 1999, 2001; Shim et al., 1997). Furthermore, studies have provided functional evidence that c-Myc increases glucose uptake and lactate production in rat fibroblasts.
An E-box bound by c-Myc in the LDHA promoter is also recognized by the hypoxia inducible transcription factor HIF1. HIF1 activity increases as the physiological response of cells to hypoxia, in which essentially all of the glycolytic enzyme genes are activated by HIF1 through cis-elements that resemble E-boxes (Dang and Semenza, 1999). Normal cells cease to proliferate in response to hypoxia, and the mechanism by which hypoxic cells cease to proliferate involves activation of the cyclin-dependent kinase inhibitor p27 (Gardner et al., 2001). While HIF1 mediates the metabolic response of cells to hypoxia, c-Myc may allow certain tumor cells to adapt to hypoxia through direct activation of glycolysis. Although induction of glycolysis may be a dominant metabolic effect of c-Myc, c-Myc responsive genes, cyclophilin, VDAC, cytochrome c, and heme oxygenase 1, involved in mitochondrial function and presumably mitochondrial respiration have also been identified (Coller et al., 2000; Guo et al., 2000). Hence, it may speculated that c-Myc regulates both glycolysis and mitochondrial respiration, although the balance between oxygen-dependent and oxygen-independent metabolism may be dictated by specific cell types.
Other Myc responsive genes, such as Ferritin H, IRP-1, and IRP-2, reflect the cellular demand in iron metabolism, and other genes reflect requirements for the synthesis of DNA, RNA and other macromolecules (Wu et al., 1999b). For example, the induction of carbamoyl phosphate synthase (CAD), dihydrofolate reductase, inosine-5′-monphosphate dehydrogenase, ODC, and thymidine kinase by c-Myc suggests a coupling between energy metabolism and the increase in DNA synthesis (Bello-Fernandez et al., 1993; Mai and Jalava, 1994; Miltenberger et al., 1995; Pusch et al., 1997b; Schuhmacher et al., 2001). The connection between c-myc and the potential regulation of a large variety of metabolic genes suggests that c-Myc is a key integrator of cell mass accumulation, energy metabolism and cell proliferation.
Although Myc function has not been clarified from murine knock-out experiments, in which c-myc null embryos die at post-coital day 10.5, it is clear that other Myc family members, such as N-myc, are insufficient for normal development (Davis et al., 1993). In contrast to c-myc, homozygous deletion of L-myc does not affect development (Hatton et al., 1996). Similar to c-myc, however, homozygous deletion of N-myc also results of embryonic lethality (Stanton et al., 1992). The embryonic lethality seen in c-myc null embryos suggests that either N-myc is unable to substitute for c-myc or that temporal and spatial regulation of c-myc expression is required in the embryo. To test this hypothesis, N-myc coding sequence has been knocked into the c-myc locus under the regulation of c-myc regulatory sequences (Malynn et al., 2000). Intriguingly, it appears that N-Myc is fully capable of substituting for c-Myc. This experiment specifically indicates that N-Myc protein could substitute for c-Myc during embryogenesis.
Homozygous deletion of c-myc in an immortalized Rat1 fibroblast cell line and the use of conditional myc knock-out animals have provide significant insights into c-Myc's role in cellular proliferation (de Alboran et al., 2001; Mateyak et al., 1997). Removal of c-Myc results in a marked prolonged doubling time. In the case of Rat1 fibroblasts, the doubling time increases from about 16 h to about 50 h without c-Myc. The myc null fibroblasts, which do not express any other Myc family proteins, display prolonged G1 and G2 phases with a conserved S phase length. In primary murine fibroblasts rendered deficient of c-Myc through Cre recombinase removal of floxed myc alleles, the doubling time increases from about 20 h to over 200 h (de Alboran et al., 2001). It is notable that viral transduction of Cre recombinase itself reduced growth rate. However, the extended doubling time resulting from the removal of c-myc was associated with an apparent G1 delay. These studies provide the genetic evidence that while c-Myc is not required for cell proliferation per se, it is required for the speed at which cells progress through the cell cycle.
The effect of ectopic c-Myc on the cell cycle machinery is context dependent. In particular, studies performed with immortalized cell lines have reached conclusions that are likely to be confounded by the fact that immortalized cell lines tend to have defects in cell cycle checkpoints. In contrast to immortalized cell lines, overexpression of c-Myc in primary cells or in vivo results in either cell cycle arrest or apoptosis, which is thought to be one mechanism by which organisms defend against neoplastic cells arising from deregulated oncogenes. Ectopic expression of c-Myc in murine fibroblasts result in the activation of p53 and p19/ARF, although the effect on ARF is likely to be indirect (Reisman et al., 1993; Zindy et al., 1998). Increased ARF sequesters MDM2, which in turn releases p53 to induce its growth inhibitory or apoptosis promoting activities. Likewise, it is shown that induction of ectopic c-Myc in an in vivo model is insufficient for cell cycle progression unless a G2 checkpoint is eliminated (Felsher et al., 2000). Hence, the dominant effect of ectopic c-Myc in the primary, non-immortalized cell is the activation of cell cycle arrest.
The regulation of normal cell proliferation by c-Myc involves several key components of the G1-S regulatory molecules (Figure 6). Evidence to date suggest several general features that may reflect the normal role of c-Myc in physiological cell proliferation. The cyclin-dependent kinase inhibitor, p27, appears to be a critical target of c-Myc (Amati et al., 1998). In Rat1 cells nullizygous for c-myc, p27 levels are elevated (Mateyak et al., 1997). Likewise, p27 levels are greatly increased in primary fibroblasts or lymphocytes that have been rendered nullizygous for c-myc (de Alboran et al., 2001). However, the inhibitor, p21, is not increased. C-Myc reduces p27 levels or activity through several mechanisms. Induction of cyclin D2 and CDK4 by c-Myc may sequester p27 from cyclin E/CDK2 into the cyclin D2/CDK4 complex although studies have also suggested that phosphorylation of p27 causes its release from cyclin E/CDK2 (Bouchard et al., 1999; Muller et al., 1997; Perez-Roger et al., 1999; Vlach et al., 1996). Additionally, induction of a component of the ubiquitin conjugating complex, CUL1, is proposed to mediate p27 degradation (O'Hagan et al., 2000a). Either or both of these mechanisms may contribute to the decrease in a critical cyclin kinase inhibitor. c-Myc may also cause a decrease in p21 expression, although this effect may be context dependent (Claassen and Hann, 2000; Coller et al., 2000). That is, depending on whether c-Myc is ectopically expressed in non-immortalized or immortalized human breast epithelial cells, p21 can either be induced or repressed (E Emison and C Dang, unpublished observation).
The activation of CDK4 by Myc could result in the phosphorylation of Rb, which results in the activation of E2F (Hermeking et al., 2000). E2F, in turn, is capable of activating cyclin E and other genes required for S phase entry. Myc also directly induces Id2; Id2 is able to inactivate Rb through direct binding (Lasorella et al., 2000). Thus, Myc may inactivate Rb through several mechanisms, but in addition Myc circumvents Rb control as well (Alevizopoulos et al., 1997). c-Myc appears to activate cyclin E and E2F2 directly and perhaps provide a means for bypassing Rb (Beier et al., 2000). E2F2 could in turn activate cyclin E in a positive feed-forward loop that pushes cells from G1 into S phase. Presumably, cyclin E and E2F2 (or other E2Fs) activates cyclin A, which is essential for S phase. Several genes involved in DNA replication such as MCM4, ORC1, and replication factor 4C may also be targeted by c-Myc (Coller et al., 2000; Schuhmacher et al., 2001). The ability of c-Myc to suppress growth arresting genes, such as gas1, GADD34, GADD45, and GADD153 is also likely to contribute to its growth promoting activities in cells that lack normal checkpoints (Amundson et al., 1998; Lee et al., 1997).
Although myc nullizygous rat fibroblasts display a prolonged G2 phase, the role of c-Myc in G2 is less clear and has not been directly studied (Mateyak et al., 1997, 1999). It may be speculated, however, that the lack of Myc's repression of p27 could well account for a prolonged G2 in cells lacking c-Myc. The prolonged G2 is not apparent in murine embryo fibroblasts lacking c-Myc, and hence whether the G2 delay in the absence of c-Myc is cell type dependent is not known.
Ectopic expression of c-Myc results in deregulated entry into S phase as well as sensitizing cells to various apoptotic stimuli (Askew et al., 1991). Ectopic Myc expression is sufficient to cause S phase entry in growth factor deprived immortalized fibroblasts (Eilers et al., 1991). Ectopic c-Myc induces cyclin E expression in early G1, in contrast the expression of endogenous Myc that is followed by cyclin E expression in late G1 (Perez-Roger et al., 1997; Pusch et al., 1997a). Despite this early expression of cyclin E, one study demonstrates that cells must attain a critical cell mass for entry into S phase (Pusch et al., 1997a). CDK2 activity is activated by ectopic c-Myc and it appears to drive cells prematurely into S phase, specifically when cells are treated with microtubule inhibitors or X-irradiated (Li and Dang, 1999; Yin et al., 1999). This ability may underlie the observations that c-Myc is able to induce genomic instability. Whether this instability also reflects the suppression of the mitotic checkpoint by c-Myc is not yet established.
When cells are deprived of growth factors (serum), they withdraw from the cell cycle and have diminished c-myc expression. In contrast, cells that express ectopic c-Myc are triggered to release cytochrome c from the mitochondria with serum-deprivation, culminating in the activation of caspases and apoptosis (Juin et al., 1999). What are the target genes of c-Myc that sensitize cells to apoptotic triggers? Both ornithine decarboxylase and LDHA have been shown to sensitize cells to serum-deprivation and glucose-deprivation induced apoptosis, respectively (Packham and Cleveland, 1994; Shim et al., 1998). Serum and glucose deprivation represent distinct stimuli since overexpression of LDHA sensitizes cells to glucose, but not serum, deprivation. Although these genes, when ectopically expressed, predispose cells to apoptosis; the molecular underpinnings that trigger a central death pathway remains poorly understood.
Myc target genes that regulate the cell cycle, such as p53 and cdc25A, have been implicated in Myc-induced apoptosis (Galaktionov et al., 1996; Reisman et al., 1993). However, the roles of p53 and cdc25A in Myc-induced apoptosis may be cell line or context dependent. Myc responsive genes involved in mitochondrial function such as cyclophilin, HSP70A and Mer5 (thioredoxin-dependent peroxidase) may also affect apoptosis; however, their roles have not been reported (Coller et al., 2000; Guo et al., 2000; Lewis et al., 1997). Bax, the antagonist of Bcl2 or Bcl-xL, has also been reported as a Myc target gene, although the results have not been confirmed by independent studies (Mitchell et al., 2000). Evidence for c-Myc activation of upstream events in the apoptotic cascade, such as activation of FasL and Fas, have been provided by several independent studies. In one study c-Myc appears to activate the Fas pathway in fibroblasts overexpressing Myc (Hueber et al., 1997). Although a separate study with FADD (a mediator of Fas apoptosis) knockout cells suggests that c-Myc induce apoptosis independently of the Fas pathway, a recent study with lymphocytes containing conditional myc knockout demonstrate that myc null lymphocytes are unable to express Fas or FasL (de Alboran et al., 2001). Furthermore, the FasL gene appears to be directly activated by c-Myc through a non-canonical Myc binding site (Kasibhatla et al., 2000). DNA microarray experiments have identified other potential Myc target genes involved in apoptosis (TRAP1, ANT, API2), although definitive studies are lacking (Guo et al., 2000; O'Hagan et al., 2000b; Schuhmacher et al., 2001).
In aggregate, several studies support the role for Fas and FasL in Myc-induced apoptosis. It is also notable that Fas-mediated apoptosis is coupled to glucose transport (Berridge et al., 1996). That is, upon FasL activation glucose transport dramatically decreases and conversely, enhancement of glucose uptake abrogates IL3-deprivation induced apoptosis (Kan et al., 1994). Although the molecular details of how Myc triggers apoptosis remain to be delineated, the understanding of the links between Myc overexpression, mitochondrial function, and glycolysis will be necessary for better appreciation of the mechanisms underlying c-Myc induced apoptosis.
Cell adhesion and differentiation
A hallmark of cellular differentiation is the expression of specific cell surface molecules that mediate cell–cell and cell–extracellular matrix interactions. Most adherent and even non-adherent cells rely on cell–cell or cell–matrix signals for mitogenesis, such that normal cells rendered non-adherent stop proliferating. Normal cells also recognize external cues and undergo contact inhibited growth when cells are confluent. This is in contrast to transformed cells that tend to proliferate independently of external cellular cues. C-myc expression in lymphocytes is associated with the decreased expression of LFA-1 that participates in homotypic interaction between lymphocytes (Inghirami et al., 1990). Decreased LFA-1 expression occurs in many Burkitt's lymphomas and hence suggests that deregulated growth could in part be coupled to loss of LFA-1.
Other cellular adhesive molecules are down-regulated by c-Myc either through transcriptional mechanisms or through the induction of metalloproteinases. Collagens have been repeatedly reported to be down-regulated by c-Myc (Yang et al., 1991). In one study, it is proposed that c-Myc interferes with the the regulation of collagen genes by nuclear factor 1 (CCAAT/NF1) (Yang et al., 1993). This is similar to the proposed mechanism for the down-regulation of the PDGFβ receptor gene by c-Myc (Oster et al., 2000). To date, however, mechanistic studies to determine whether collagens could inhibit Myc-mediated tumorigenesis have not been performed. Other potential repressed targets such as fibronectin have also been reported (Coller et al., 2000). One study demonstrated that Myc reduces α3 mRNA expression in a small cell lung cancer cells line, leads to a decrease in α3β1 integrin receptor and enhances anchorage independent growth (Barr et al., 1998). Restoration of α3 expression neither reduces the growth rate nor c-Myc expression, but it dramatically blocks anchorage-independent growth. This study provides evidence that reduction of a specific adhesion receptor by c-Myc enhances anchorage-independent growth. It is further supported by an independent study demonstrating that restoration of β1 expression in fibroblasts lacking β1 causes a significant reduction in the size of myc and ras induced tumors (Brakebusch et al., 1999). These studies together suggest that down-regulation of adhesion molecules by c-Myc may be required for neoplastic transformation.
Thrombospondin is a matrix-associated protein with a negative regulatory role in angiogenesis. It is repressed by c-myc, probably through specific post-transcriptional mechanisms (Janz et al., 2000). While it is clear that c-myc mediates tumorigenesis, whether c-Myc induces neovascularization is not yet clear (Ngo et al., 2000). In vivo models suggest that c-Myc induces vascular endothelial growth factor VEGF (Pelengaris et al., 1999). However, in several cell culture models, ectopic Myc expression caused a decrease in VEGF mRNA, although whether cell type contributes to the differences is unknown (Barr et al., 2000). It is likely that the regulation of angiogenesis by c-Myc differs depending on the process, whether it is embryogenesis, wound repair or tumorigenesis. It is possible that during embryogenesis or perhaps wound repair, physiological expression of Myc directly couples with angiogenesis; in contrast, ectopic overexpression of c-Myc could result in the suppression of angiogenesis as an additional mechanism to protect organisms against tumorigenesis.
c-Myc has long been shown to antagonize differentiation programs of several cell culture models including myocyte and adipocyte differentiation. The failure of cells expressing ectopic Myc to withdraw from the cell cycle is thought to prevent them from expressing their differentiated phenotype. The concept that withdrawal from the cell cycle is required for cellular differentiation is now contested by the observations that continued Myc expression is required for differentiation of keratinocytes (Gandarillas et al., 2000). Furthermore, the observation that B-cells with ectopic c-myc expression in vivo show limited differentiation suggests that c-myc expression and differentiation are not necessarily mutually exclusive.
Senescence and immortality
Some of the landmark conceptual shifts stemmed from the study of single versus multiple oncogenes in primary cells. The ability of Myc and Ras to transform primary rodent cells provided an early conceptual framework for our understanding of multistep tumorigenesis which was later modified by the recognition of tumor suppressors. The oncogenes were thought to cooperate through their respective complementary ability to drive cell proliferation (Land et al., 1986). That is, in the case of Myc and Ras, it was thought that Myc is able to immortalize primary cells and that Ras provides additional oncogenic cues to drive cells into full tumorigenic potential. This simple view overlooked the early events of ectopic c-myc expression in primary cells (Evan et al., 1992; Zindy et al., 1998). As it is now known and discussed above, c-Myc over-expression in primary cells tends to result in cell cycle arrest or apoptosis. Only cells that survive this crisis and massive exodus by death become immortalized by c-Myc (Zindy et al., 1998). The recognition that c-Myc activates telomerase activity and directly activates the gene encoding the reverse transcriptase subunit, TERT, suggests a possible mechanism for c-Myc mediated immortalization (Greenberg et al., 1999; Wang et al., 1998; Wu et al., 1999a). Yet Myc and Ras together are unable to transform human primary cells (Hahn et al., 1999). In fact, human primary cells require ectopic expression of hTERT for neoplastic transformation by activated Ras and SV40T antigen. The difference in the susceptibility of rodent versus human cells to Myc and Ras transformation may reflect the propensity for rodent cells to maintain telomerase activity and have much longer telomeres. Thus, the role of c-Myc in regulating hTERT physiologically or in tumorigenesis remains to be established for human tissues.
In contrast to c-Myc, activated Ras expression alone in primary cells results in senescence rather than cell cycle arrest per se (Serrano et al., 1997). The potential ability of c-Myc to maintain telomerase activity could counter the senescent effect of Ras. Ras is now known to suppress apoptosis through AKT and PI3K, which is likely to suppress the apoptotic effects of c-Myc (Rohn et al., 1998). These recent observations offer a new view, which suggests that complementation between oncogenes includes both complementary growth promoting effects as well as cross-suppression of normal safeguard mechanisms elicited by overactive oncogenes (Figure 7).
The spectrum of normal and abnormal myc function
Regulated c-myc expression is required for normal embryogenesis and cellular proliferation, as is reflected by the phenotypes of embryos that are homozygously depleted of c-myc or primary cells rendered deficient of c-myc. To reach an understanding of normal c-myc function, reagents derived from these animals will have to be judiciously studied to identify normal c-myc target genes. The use of cell lines with inactivated c-myc alleles have yielded novel insights; however, it is likely that some compensatory mechanisms must be at play to account for a large difference between the doubling time of primary cells deprived of c-myc versus that of an immortalized rodent cell line. The differences in growth rates between wild-type and myc null cells will pose a challenge in the interpretation of any studies that rely on growing cells. The experimental manipulation of cells through serum deprivation and re-stimulation to trigger mitogenesis remains a feasible model to study target genes of c-Myc. Perhaps with the use of DNA microarrays and chromatin immunoprecipitation assays, a better understanding of physiological Myc target genes will emerge.
Deregulated or abnormal function of c-Myc as depicted by chromosomal translocations in B-cell malignancies is likely to elicit a distinctly different subset of Myc target genes. To date, one study attempted to address this issue using isogenic wild-type, myc null, and wild-type cells plus ectopic myc and DNA microarray technology (Guo et al., 2000). There appears to be a distinct subset of c-Myc responsive genes that are more readily detected between ectopic myc expressing wild-type cells and control wild-type cells versus myc null cells and control wild-type cells. In summary, while c-myc is not required for cell proliferation as is evident from the proliferation of myc null cells, the pleiotropic transcriptional effect of c-Myc suggests that it is a central integrator and acceleration of physiological cell growth, proliferation and cellular metabolism. Ectopic expression of c-Myc most likely contributes to tumorigenesis through both temporally disordered expression of physiological target genes and the abnormal expression of non-physiological target genes that are not regulated by endogenous c-myc. With advances in technologies that enable investigations to incorporate spatial and temporal regulation of gene expression, understanding of normal and abnormal c-Myc function should be rapidly forthcoming.
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Supported by National Institutes of Health Grant CA69322 (LM Boxer), CA51497 (CV Dang) and CA57431 (CV Dang). We thank E Emison, L Gardner, LA Lee and D Wechsler for comments.
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Boxer, L., Dang, C. Translocations involving c-myc and c-myc function. Oncogene 20, 5595–5610 (2001). https://doi.org/10.1038/sj.onc.1204595
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