Proteins of the Myc Network: Essential Regulators of Cell Growth and Differentiation

Proteins of the Myc Network: Essential Regulators of Cell Growth and Differentiation Marie Henriksson and Bernhard Liischer Institute for Molecular Biology, Hannover Medical School, 0-3062.3 Hannover, Germany

I. Introduction

11. The myc Genes and Functional Domains of the Myc Proteins 111.

IV.

V.

VI.

VII.

A. The myc Gene Family B. Myc Structure The Myc Network A. The Myc Dimerization Partner Max B. M a x Association with Mad Family Proteins C. DNA Binding of Max-Containing Complexes D. Myc as a Transcription Factor E. Proteins Interacting with c-Myc Myc Target Genes A. Indirect and Direct Target Genes B. a-Prothymosin C. Ornithine Decarboxylase D. p53 E. ECA39 F. cad G. Genes Encoding Cell Cycle Regulators The Role of Myc Network Proteins in the Cell Cycle and during Differentiation A. Myc in Cell Proliferation B. Expression Pattern of the myc Family Genes C. Effects of c-Myc on Differentiation D. Targeted Disruption of the c- or N-myc Genes E. Effects of Max on Differentiation F. Expression Pattern of the mad Family Genes Myc-Mediated Apoptosis A. Overexpression of c-Myc Triggers Apoptosis B. Regulation of Myc-Induced Apoptosis C . How Does c-Myc Modulate Apoptosis? Effects of Myc Network Proteins on Transformation A. Alterations of c-myc in Tumors B. Tumorigenesis in c-myc Transgenic Mice C. Cooperating Oncogenes in c-myc Transgenic Mice

Advances in CANCER RESEARCH, Val. 68 Copyright 0 1996 hy Academic Press, Inc. All rights of reproduction in any form reserved.

I10

Marie Henriksson and Bernhard Luscher

D. Assay Systems for in Vitro Transfomation E. Analysis of c-Myc Transformation in Vitro F. Influence of Mad Family Proteins on Transformation by Myc and Other Oncoproteins G. Chromosomal Localizations of max, mad, mxil, mad3, and mad4: Do These Genes Encode Tumor Suppressors? V111. Future Aspects References

1. INTRODUCTION The development and homeostasis of multicellular organisms requires the precise regulation of cell growth, differentiation, and death. Loss of balance between these processes can lead to numerous malfunctions including the development of tumors. Multiple layers of controls have been established to ensure proper development and homeostasis. Consistent with this is the observation that several mutations have to be accumulated within a cell for it to escape growth control imposed by the organism and to develop into a malignant tumor cell. The identification of oncogenes and their protein products, first as sequences transduced in retroviruses and subsequently as the corresponding normal cellular homologues, defined an important class of proteins playing a critical role in cell growth control. Later it was discovered that these normal genes or protooncogenes were activated in certain tumors and appeared to be involved in the onset as well as the progression of the disease. The cellular protooncogenes can be activated by structural and functional alterations, such as point mutations, truncations, chromosomal rearrangements, gene amplifications, or proviral insertions. The activated protooncogenes involved in tumorigenesis are called cellular oncogenes. More than 70 oncogenes have been identified to date and, although the precise function of many protooncogenes is still unclear, several studies indicate that many of their protein products participate in signal transduction pathways induced by growth and differentiation factors. On the basis of their cellular location and functional properties they are classified as growth factors, growth factor receptors, signal transducers, protein kinases, or transcription factors. The protooncogenes are therefore likely to play an important role in growth control and thereby in determining cell fate. In addition to these classes of protooncogenes, recent evidence indicates that several cell cycle regulators, such as certain cyclins and E2F transcription factors, also possess tumorigenic potential. These findings support the concept that cell cycle regulators are downstream of signal transduction cascades involved in

Myc Proteins

111

growth control and can provide dominant growth stimulatory signals upon activation. Tumor suppressor genes, on the other hand, encode proteins that may counterbalance the effect of protooncogenes. In a normal cell they modulate growth-promoting signals, transcription, DNA repair, and DNA replication. Like the protooncoproteins they act at pivotal points during the cell cycle in order to maintain homeostasis. Loss of function of these genes by deletion or mutation also contributes to tumor development. c-myc is one of the most widely studied protooncogenes. It is the best characterized member of the rnyc family of protooncogenes which encode short-lived nuclear phosphoproteins. The strong interest in c-myc derives from the early descriptions of a number of human tumors with genetic alterations at the c-myc locus. Data from various experimental systems then demonstrated that the activation of c-myc provides a selective advantage important in tumor development. With this knowledge both the c-myc gene as well as its protein product, c-Myc, became the focus of intense research. It was of prime interest to understand the regulation of c-myc expression as well as the function of the protein because the expectation was that this information would provide insight into tumor formation. In the 1980s a number of suggestions were made concerning the function of c-Myc, including a role for this protein in the structural organization of the nucleus, in mRNA splicing, in DNA replication, and in gene transcription. Evidence for all these has been obtained but at present it appears that c-Myc’s function is best explained through its activity as a transcription factor. The identification of the Myc partner, Max, in 1991 and the subsequent realization that this protein is the essential dimeric partner for all known c-Myc functions was a major boost to the field and led to a number of very interesting observations and findings. In this review we will focus on these new developments. The regulation of c-myc gene transcription and the mechanisms of rnyc activation in malignancies will not be discussed in detail and have been covered in excellent reviews (DePinho et al., 1991; Spencer and Groudine, 1991; Marcu et al., 1992). We will concentrate on C-MYC’S role as a transcription factor in the regulation of cell growth, apoptosis, and transformation. Furthermore, we will summarize data on c-Myc-interacting proteins and their role in modulating c-Myc function as well as novel partners of Max, Mad (now also referred to as Madl), Mxil, Mad 3, and Mad 4 which have been identified. The most exciting recent findings suggest that the Myc network not only includes protooncoproteins (c-, N-, and L-Myc), but with the Mad family proteins, also potential tumor suppressors. This together with the fact that Myc proteins as well as Max are essential, as deduced from homozygous disruption of the genes in mice, places the Myc network in a central position in the regulation of cell growth and homeostasis.

112

Marie Henriksson and Bernhard Luscher

11. THE myc GENES AND FUNCTIONAL DOMAINS OF THE Myc PROTEINS

A. The myc G e n e Family Genes that have been generated by duplication of and divergence from an ancestral gene(s) are grouped into families. The myc family of protooncogenes has most likely arisen through such duplications. It currently consists of three well-characterized members; c-myc, N-myc, and L-myc. Two additional genes, B-myc and S-myc, have been identified only in rodents. The c-, N-, and L-myc genes share similar genomic organization and the corresponding proteins contain several regions of high sequence homology. These appear also functionally related as mentioned below. 1 . THE V-myc G E N E

The myc gene was originally identified as the oncogene of the MC29 avian leukemia virus (ALV) (Sheiness et al., 1978; Sheiness and Bishop, 1979). Four other acute ALVs carrying the v-myc oncogene, MH2, CMII, OK-10, and FH3, have been isolated (Bister and Jansen, 1986; Chen et al., 1989). These retroviruses induce carcinomas, endotheliomas, and sarcomas in addition to the leukemic disorder myelocytomatosis (hence myc) in susceptible birds and transform fibroblasts and macrophages in tissue culture (reviewed in Graf and Beug, 1978).

2. THE

C-,

N-, L-, S - , AND B - W ~ GENES C

The c-myc gene was first isolated as the chicken cellular homologue of v-myc (Vennstrhm et al., 1982), and subsequently the human, mouse, and rat c-myc genes were cloned and characterized (Dalla-Favera et al., 1982; Stanton et al., 1984; Hayashi et al., 1987).The c-myc gene is evolutionarily conserved and has also been cloned from trout, frog (Xenopus laevis), zebra fish, and the nonvertebrate sea star Asterias vulgaris (King et al., 1986; Taylor et al., 1986; van Beneden et al., 1986; Walker et al., 1992; SchreiberAgus et al., 1993). However, no c-myc homologues have been discovered in Drosophila, Caenorhabditis elegans, or in yeast. The N-myc and L-myc genes were originally identified as amplified mycrelated genes in human neuroblastoma and small cell lung cancer, respectively (Kohl et al., 1983; Schwab et al., 1983; Nau et al., 1985). The three myc genes share the same general topography with the main open reading frame retained within the second and third exons. There are two highly

Myc Proteins

I I3

homologous regions encoded within exon 2 and three within exon 3 of all three rnyc genes. These so-called myc boxes are also evolutionarily conserved between myc genes from different species. L-myc is more distantly related to the other two members although it shares organizational features and several myc boxes. S-myc and B-myc are less well-characterized members of the rnyc family. S-myc was isolated from a rat genomic library using v-myc as a probe (Sugiyama et al., 1989). It exhibits high homology to the N-myc second and third exons but lacks the intron, thus its coding region comprises a single exon. B-myc has been cloned from both rat genomic as well as cDNA libraries by homology to the second exon of c-myc and lacks sequences corresponding to the third exon entirely (Ingvarsson et al., 1988; Asker et al., 1995). The genomic structures of both S- and B-myc have diverged significantly from the other members of the family and the promoters are ill defined at present. S-myc expression has only been detected in rat embryo chondrocytes at extremely low levels, whereas B-myc expression was observed in a number of tissues with highest levels in brain (Ingvarsson et al., 1988; Asai et al., 1994). To date, no endogenous proteins have been identified for either S- or B-myc. It remains to be determined if these encode functional genes or if they represent pseudogenes. The Myc proteins can substitute for each other in certain situations. We will focus our discussion on c-Myc and only mention the other Myc family members in cases in which there are differences between the proteins, for example, in expression pattern during embryogenesis and in adult tissues.

B. Myc Structure

I . THE C-Myc PROTEIN The human c-myc gene encodes two polypeptides with apparent molecular weights of 64 and 67 kDa, respectively (Hann et al., 1988). Translation of the larger protein (Myc-1 or p67) is initiated at a cryptic start codon at the end of exon 1, whereas the smaller protein (Myc-2 or p64) is produced from an ATG start codon in c-myc exon 2 (Hann et al., 1988). p64, which represents the major translational product, consists of 439 amino acids and p67 has an N-terminal extension of 14 amino acids. The c-Myc proteins are nuclear phosphoproteins with a half-life of about 20-30 min (see Luscher and Eisenman, 1990). Figure 1 shows a schematic diagram of the c-Myc structure and a delineation of domains that play key roles in different functional activities. These domains will be described below and their importance for the respective biological function will be discussed in the following sections.

114

Marie Henriksson and Bernhard Luscher

C-MYC -14 1

439

100 amino adds

Transcriptional regulation Transformation S-Phase Induction Apoptosis Inhibition of Differentiation p107 TBP a-tubulin

Max YY-1

Protein-Protein Interactions

AP-2

Fig. 1 Structural and functional domains of c-Myc. c-Myc harbors a transcriptional activation domain (TAD; amino acids 1-143), a basic region (b; amino acids 355-368), a helix-loop-helix motif (HLH; amino acids 368-410), and a leucine zipper domain (Zip; amino acids 41 1-439). The HLHZip domains mediate protein-protein interaction while the b region specifies DNA binding. The shaded boxes within the TAD represent Myc Box 1 ( I ; amino acids 45-63) and Myc Box 11 (11; amino acids 129-141), respectively, two conserved regions found in all Myc family members. Residues 320-328 specify the main nuclear localization signal (NLS).Amino acids 242-261 contain a highly acidic region (A).The locations of the major in vivo phosphorylation sites are indicated with P. Myc-1 (p67) contains 14 additional residues at its amino terminus, due to a CUG initiation codon in exon 1 , compared to Myc-2 (p64) which is initiated at the first AUG in exon 2. Myc domains linked t o function in different assays as well as involved in protein-protein interactions are indicated.

2. PROTEIN AND DNA INTERACTION MOTIFS The prediction that the Myc proteins could dimerize and/or confer specific DNA binding emanated from the recognition of homologies between the carboxy-terminal domain of c-Myc and structural features previously identified in transcriptional regulators. These similarities fall into three classes: the helix-loop-helix (HLH) and leucine zipper (Zip) motifs, which can promote protein-protein interaction, and the basic region (b), which mediates sequence-specific DNA binding. Transcription factors harboring these motifs are referred to as bHLH, bZip, or bHLHZip proteins, respectively. The Myc proteins were shown to contain a bHLH domain as well as a Zip region (see Fig. 1). The bHLH class of transcriptional regulators harbors a basic region followed by two amphipathic a-helices of about 15 amino acids joined by an intervening loop. Dimerization occurs via hydrophobic residues that line up on one side of each of the amphipathic a-helixes positioning the basic region

Myc Proteins

I15

in a favorable configuration for sequence-specific DNA binding (Murre et al., 1989). Heterodimer formation between different bHLH proteins generates a large potential for regulatory diversity. Furthermore, the bHLH proteins share the ability to recognize a common DNA sequence referred to as the E-box (CANNTG), thereby providing opportunities for regulation through competition for DNA binding. The myogenic control proteins MyoD and myogenin, the immunoglobulin K enhancer binding proteins, E l 2 and E47, as well as the Drosophila differentiation factors, achaetescute, daughterless, and twist, are examples of members of the bHLH family (Murre et al., 1989; Olson, 1990). The HLH protein Id, which can heterodimerize with E12, E47, or MyoD, lacks the basic DNA-binding motif (Benezra et al., 1990). To date, there are four different Id proteins identified (see Riechmann et al., 1994). The product of the Drosophila extramacrochaetae locus, Emc, is another HLH protein lacking the basic region (Ellis et al., 1990; Garell and Modolell, 1990). Both the Id proteins and Emc function in an antagonistic manner since heterodimerization with these proteins results in DNA binding incompetent complexes. In addition to these negatively acting proteins there are bHLH proteins that contain a proline residue in their basic region. Examples of such proteins are the Drosophila hairy and Enhancer of split gene products and the rat proteins HES-1 and HES-3 (Ingham et al., 1985; Klambt et al., 1989; Sasai et al., 1992). Proteins in this subgroup have an altered DNAbinding capacity in that they do not bind the E-box motif but rather the socalled N-box motif (CACNAG) (Sasai et ul., 1992; Tietze et ul., 1992). The bZip proteins are characterized by a basic domain followed by a 30to 40-amino-acid-long a-helix containing four or five leucine residues or other hydrophobic amino acids interspersed at every seventh position (Landschulz et al., 1988).This heptad repeat mediates dimerization and juxtaposition of two basic regions to form the DNA binding site. The contact surface consists of the hydrophobic amino acids that are directed toward one side of the helix as well as additional hydrophobic amino acids at the + 4 position of each leucine, the alternate hydrophobic repeat (Alber, 1992).The stability of the dimer results from the packing of the leucines and nonpolar residues and from a limited number of intra- and interhelical salt bridges (O’Shea et al., 1991). The zippers form a parallel coiled coil that is positioned in a perpendicular orientation to the basic regions which then can contact a specific recognition site in the DNA (O’Shea et al., 1991; Ellenberger, 1992). The specificity of complex formation is determined by the individual Zip domains; therefore, not every leucine zipper region can interact with every other (Baxevanis and Vinson, 1993). The positioning of additional hydrophobic as well as charged amino acids determines the possible combination of the interaction. Examples of transcriptional activators belonging to the bZip class are C/EBP, Fos, Jun, and the yeast protein GCN4 (Landschulz et

I I6

Marie Henriksson and Bernhard Luscher

al., 1988). Another bZip protein, Chop-10, contains two prolines in its basic region and functions as a dominant negative inhibitor of C/EBP by sequestering it into a heterodimer that is unable to bind DNA (Ron and Habener, 1992). The Myc family members possess both a H L H and a Zip in a contiguous arrangement preceded by a basic region and are thus referred to as bHLHZip proteins. Other proteins in this category include USF, AP4, TFE3, and TFEB (Beckmann et al., 1990; Carr and Sharp, 1990; Gregor et al., 1990; H u et al., 1990) as well as the proteins involved in the Myc network, i.e., Max, and the Mad family members (see Section 111,B). Extensive deletion and insertion mutagenesis of the c-myc gene has shown that the bHLHZip region is critical for Myc function (Stone et al., 1987; Crouch et al., 1990; Freytag et al., 1990; Penn et al., 1990a; Smith et al., 1990; Evan et al., 1992; Kretzner et al., 1992; Amin et al., 1993; Gu et al., 1993; Goruppi et al., 1994; see Fig. 1) suggesting that Myc exerts its function through specific DNA binding. Myc cannot form homodimers under physiological conditions and Max is, so far, its exclusive dimerization partner interacting through the HLHZip domain. Max, on the other hand, can homodimerize as well as heterodimerize with other bHLHZip proteins of the M a d family (see Section 111,B). The presence of two dimerization motifs is intriguing and raises the interesting possibility that the Zip and the HLH could mediate complex formation with different sets of factors which could either be simultaneous o r mutually exclusive. Alternatively, the presence of two distinct dimerization domains could ensure a high level of discrimination with respect to partner. This latter model is supported by experimental data in which the TFE3 HLH was fused to the Zip region of USF and by the finding that the Zip functions in cooperation with the HLH to stabilize protein-protein interactions and to establish dimerization specificity (Beckmann and Kadesch, 1991). For USF, it has been demonstrated that the Zip motif allows the formation of a homotetramer that simultaneously can bind two spatially distinct DNA recognition sites indicating a role for the HLHZip domain in DNA looping (Ferrk-D’Amari et al., 1994). Furthermore, the presence of two motifs allows for additional dimerization specificity by variation of the distance between the Zip and the HLH (Baxevanis and Vinson, 1993). All these possible variations are at present hypothetical for c-Myc but it is appealing to speculate that Myc might associate with additional partner proteins besides Max. Such heterodimeric complexes may perform novel functions currently not anticipated for c-Myc or other Myc family members. The structural homology of c-Myc with USF and TFE3, which both bind the E-box sequence, CANNTG, led to the prediction that the basic domain of c-Myc would also recognize an E-box element. This was verified in studies in which either a bacterially expressed c-Myc fragment o r a chimera con-

Myc Proteins

I I7

taining the c-Myc basic domain linked to the E12-HLH domain were shown to bind the palindromic core sequence CACGTG (Blackwell et al., 1990; Prendergast and Ziff, 1991). Several other groups also reported binding of Myc to the same sequence element (Fischer et al., 1991; Halazonetis and Kandil, 1991; Kerkhoff et al., 1991). 3. THE TRANSCRIPTIONAL ACTIVATION DOMAIN

Transcription factors appear to be composed of at least two separate functional domains, one sequence-specific DNA binding domain and one domain capable of activating transcription when bound to DNA in the vicinity of a promoter. A potential transcriptional activation domain (TAD) has been mapped to the amino terminus of c-Myc using fusion proteins between c-Myc and the DNA-binding domain of the yeast transcriptional activator GAL4 (GAL4-Myc) (Kato et al., 1990). The TAD of Myc was subdivided into region A (amino acids 1-41, glutamine-rich and slightly acidic), region B (amino acids 41-103, containing 3 3 % prolines) and region C (103-143, no resemblance to any previously described transcriptional activation motif) (Kato et al., 1990). The TAD contains two Myc Boxes that are highly conserved between the Myc family proteins as well as between species. Myc Box I roughly comprises amino acids 45-63 and Myc Box I1 amino acids 129-141 (Ingvarsson, 1990; Figure 1).Besides the bHLHZip the N-terminal TAD is the second domain essential for c-Myc function. A large number of activation regions in various transcription factors have been characterized and examples of such domains are the acidic region of the herpes simplex virus VP16, the glutamine-rich domain of the human S p l protein, and the proline-rich region of the human protein CTFI/NFI (for reviews see Mitchell and Tjian, 1989; Johnson et al., 1993; Triezenberg, 1995). Mutational analysis of the TAD of herpes simplex VP16 has indicated that the minimal activation domain contains both acidic and hydrophobic residues (Cress and Triezenberg, 1991; Seipel et al., 1994). These features are likely to specify the interactions with components of the transcriptional machinery, such as the general transcription factors TBP, TFIIB, and TFIIH, as well as with TBP-associated factors (TAFs) (Stringer et al., 1990; Lin et al., 1991; Goodrich and Tjian, 1994; Xiao et al., 1994). It has even been suggested that the main common feature of the different classes of transactivators is the pattern of bulky hydrophobic residues rather than their most common amino acids (Triezenberg, 1995). Many transcription factors have in addition been shown to be dependent on coactivator o r adaptor proteins to bridge the gap between the activator and the general transcription machinery. Examples of bridging proteins are CBP, which enables transcriptional activation of CREB, Jun, and Myb, as well as the p300 protein, which

I la

Marie Henriksson and Bernhard Luscher

mediates repression by adenovirus E1A (Arias et af., 1994; Kwok et af., 1994; Arany et al., 1995; Oelgeschlager et al., 1995). If c-Myc interacts with the transcriptional machinery directly or through coactivators is presently unclear. However, binding to TBP has been shown recently, offering one possible mechanism by which c-Myc confers transcriptional activation (see Section 111,E).

4. NUCLEAR LOCALIZATION SIGNALS Two domains in the carboxy terminus of Myc have been shown to direct its targeting to and retention in the nucleus. Amino acids 320-328 comprise the main nuclear localization signal (NLS1) that induces complete nuclear localization (Dang and Lee, 1988). This region has sequence homology to the nuclear localization signals of SV40 and polyoma T antigen (Kalderon et af., 1984; Richardson et al., 1986). The second nuclear localization signal (NLS2, residues 364-374) coincides with the basic DNA-binding region and confers only a partial nuclear targeting. Whereas c- and N-Myc contain both NLSl and 2, L-Myc retains only NLS2 and has no region homologous to NLS1. Some aspects of nuclear versus cytoplasmic distribution of c-Myc will be discussed in Section V,A.

5. POST-TRANSLATIONAL MOD1FlCATlONS All the major in vivo phosphorylation sites of c-Myc have been mapped (see Fig. 1)and potential kinases identified. The ubiquitous Ser/Thr-specific kinase casein kinase I1 (CKII) phosphorylates c-Myc in vitro in the central acidic region (amino acids 240-262) and in a segment close to the basic region (amino acids 342-357) at sites that are modified in vivo (Luscher et al., 1989). Both these regions are conserved between the Myc proteins but little is known about the functional relevance of these phosphorylations (see Street et al., 1990; Blackwood et al., 1994). Mutants of MC29 v-Myc with deletions covering the acidic domain were found to have an altered tissuespecific transforming capacity (cited in Luscher et al., 1989). The evolutionarily highly conserved amino-terminal domain is also phosphorylated in vivo. At least three sites (Thr-58, Ser-62, and Ser-71) have been identified which can be phosphorylated in vitro by a number of kinases including glycogen synthase kinase 3 (GSK3), mitogen-activated protein kinase (MAP kinase), ~ 3 4 4 (CDKl), ~2 and a pl07/cyclin A/CDK complex (Alvarez et al., 1991; Henriksson et al., 1993; Lutterbach and Hann, 1994; Pulverer et al., 1994; Hoang et af., 1995). The amino-terminal phosphorylation sites are localized in a region which is part of the TAD and which is also of importance for the transforming potential of c-Myc (Sarid et al., 1987;

119

Myc Proteins

Stone et al., 1987; Kato et al., 1990). Thr-58 is believed to be an important regulatory site because it is frequently mutated in activated forms of Myc (see Section VII,E). Furthermore, mutational analysis of this site demonstrated its significance in in vitro transformation assays. Interestingly, Thr-58 has been found to be modified by glycosylation but the functional significance of this is not known (Chou et al., 1995a,b).

Ill. THE MYC NETWORK A. The Myc Dimerizatlon Partner

Max

The identification of the Myc dimerization partner Max has significantly advanced our understanding of the molecular function of c-Myc. The prediction that Myc would have a specific partner originated from the observations of heterodimer formation of other bHLH and bZip proteins as well as from the lack of Myc homodimers under physiological conditions. Max was originally identified by screening a human cDNA expression library with a radiolabeled fusion protein containing the Myc carboxy terminus (Blackwood and Eisenman, 1991). The discovery of Max facilitated the identification of the murine homologue initially called Myn (Prendergast et al., 1991). Max is a bHLHZip protein (see Fig. 2) that forms heterodimers with c-Myc, N-Myc, and L-Myc as well as homodimers with itself (Blackwood and Eisenman, 1991; Wenzel et al., 1991; Blackwood et al., 1992; Mukherjee et al., 1992). All of these complexes bind to the same E-box sequence, 5’-CACGTG and it has also been shown that Myc/Max dimers can recognize noncanonical DNA sequences (Blackwell et af., 1993). Myc and Max dimerize through their HLH and Zip motifs, while the basic region specifies DNA binding (Crouch et al., 1993; Davis and Halazonetis, 1993). Predictions from the structural motifs found in Max and the information obtained from mutational analysis complement the crystallographic structure of the Max homodimer complexed to DNA. X-ray analysis revealed a symmetrical, parallel, left-handed bundle of four a-helices (Fig. 3; FerriD’Amari et af., 1993). Each monomer contains two helices separated by a loop. The first helix consists of the basic region and helix 1, whereas the second helix is composed of helix 2 and the leucine zipper. Therefore, both a-helices represent the contiguous array of two structural elements. The homodimer is stabilized by conserved hydrophobic amino acids that are buried in the interior of the structure. Like in GCN4, the basic regions of Max revealed a-helical structures that projected from the bundle toward the

120

Marie Henriksson and Bernhard Luscher

Fig. 2 Schematic comparison of the structures of c-Myc, Max, Mad, and M x i l . Abbreviations: TAD, transcriptional activation domain; b, basic region; HLH, helix-loop-helix domain; Zip, leucine zipper motif; H R I and 2, Mad/Mxil homology region 1 and 2. H R I mediates the interaction of Mad and Mxil with mSin3. Max p21 and Max p22 differ by a 9-amino-acid insertion close to the DNA-binding region (b). Murine Mxil-WR and Mxil-SR differ by 36 amino acids including the N-terminal H R l domain.

major groove of the DNA and the second helix formed an a-helical coiled coil (Ellenberger et al., 1992; Ferrk-D’Amark et al., 1993). The first analysis indicated that Max, in contrast to Myc, has a long halflife and is constitutively expressed under diverse conditions in a number of different cell types (Berberich et al., 1992; Blackwood et al., 1992). Furthermore, its expression did not appear to change during the cell cycle o r during differentiation. However, there have been reports presenting evidence that the max gene is also regulated (see Section V,E). Max is highly conserved in the evolution of vertebrates and homologous genes have been cloned from chicken, frog, and zebra fish (King et al., 1993;

Myc Proteins

121

Fig. 3 Schematic view of the Max homodimer interacting with DNA (from Ferri-D’Amare et

al., 1993, Fig. 3b).

Schreiber-Agus et al., 1993; Sollenberger et al., 1994; Tonissen and Krieg, 1994). Transcripts exist in several alternatively spliced forms, the two major ones encode p21Max and p22Max, differing by a nine-amino acid insertion close to the basic region in p22Max (Blackwood and Eisenman, 1991; see Fig. 2). Both proteins can homodimerize as well as heterodimerize with c-Myc. In chicken only the p22 form is found (Sollenberger et al., 1994). In contrast to c-Myc, Max is a stable nuclear protein with a half-life of more than 14 hr (Blackwood et al., 1992). It has an acidic region and a NLS in its C-terminus but does not contain a TAD (Kato et al., 1992). In vivo phosphorylation sites have been identified in the acidic C-terminal domain as well as on sites close to the basic region (Berberich and Cole, 1992; Bousset

122

Marie Henriksson and Bernhard Luscher

et al., 1993; Koskinen et al., 1994). The amino-terminal sites can be phosphorylated by CKII in vitro and this phosphorylation alters the DNA-binding properties of Max homodimers and heterodimers between Max and a C-terminal fragment of c-Myc (Berberich and Cole, 1992; Bousset et al., 1993). However, no effect was observed on full-length c-Myc/Max complexes derived from eukaryotic cells, whereas the Max homodimeric complexes were still sensitive to phosphorylation (K. Bousset and B. Liischer, unpublished observation). The use of another alternative exon results in the production of the C-terminally truncated AMax proteins, p l 6 M a x and pl7Max, which, however, seem to represent a minor fraction of the total Max protein (Makela et al., 1992). Whereas the functional differences between p21 and p22 Max have not been solved in detail, p16 and p17 have been demonstrated to differ in their effect on Myc/Ras transformation compared to the p21 and p22 forms (see Section VI1,E).

B. Max Association with Mad Family Proteins The presence of Max during quiescence and differentiation, when Myc is downregulated (see Sections V,C and V,E), raised the possibility that additional proteins might exist that associate with Max. Using protein-protein interaction screens, two novel bHLHZip proteins, Mad and M x i l , were identified (Ayer et al., 1993; Zervos et al., 1993). Similarly to Myc, neither of these proteins form homodimers, however both can readily heterodimerize with Max to form DNA-binding competent complexes that recognize the same CACGTG E-box sequence as the Myc/Max heterodimers (Ayer et al., 1993; Zervos et al., 1993; Cerni et al., 1995). Three regions of homology between Mad and M x i l have been identified. In addition to the bHLHZip domains, 28 amino acids at the N-termini and 69 amino acids C-terminal to the Zip domains show extensive homology (75% and 67% identity, respectively) suggesting their functional relevance (Ayer et al., 1993; Zervos et al., 1993; Fig. 2). The N-terminal homology domain (HR1) has recently been shown to mediate interaction with mSin3A and B (Ayer et al., 1995; Schreiber-Agus et al., 1995; see below), and it is possible that the C-terminal homology region (HR2) also serves as a protein interaction domain. Analogous with Max, Mad and M x i l seem to lack an obvious TAD. Mad migrates as a doublet of 35 kDa in SDS-PAGE and has a short half-life of 15-30 min (Ayer and Eisenman, 1993). Presumably the same is true for M x i l but no data on endogenous proteins have been reported to date. Due to alternative splicing the murine mxil gene gives rise to two mRNAs which differ in their capacity to encode a 36-amino-terminal extension containing the HR1 (Schreiber-Agus et d., 1995; see Fig. 2). Recently, two novel bHLHZip proteins related to Mad (now also referred

Myc Proteins

123

to as M a d l ) and Mxil were identified and characterized (Hurlin et al., 1995a). These proteins, Mad3 and Mad4, migrate with apparent molecular weights of 29 kDa and 32 kDa, respectively, and show extensive sequence homologies to Mad and Mxil in the bHLHZip, the HR1, and the HR2 domains. These new Mad family members heterodimerize with Max to form DNA-binding competent complexes.

C. DNA Binding of Max-Containing Complexes All the dimeric complexes described previously can bind to a 5’-CACGTG consensus sequence. The Myc/Max complexes seem to exhibit a higher specific DNA binding than Max homodimers alone (Blackwood and Eisenman, 1991; Solomon et al., 1993). However, affinities of cellular complexes have not been determined due to difficulties in obtaining DNA-binding activities with full-length c-Myc. Recently, the use of extracts derived from COS-7 cells overexpressing c-Myc has demonstrated DNA-binding competent Myc/Max complexes (Bousset et al., 1995) which should enable studies on DNA-binding kinetics in the future. It has been suggested that Max homodimers are less discriminating in their DNA binding than Myc/Max heterodimers, the latter showing certain preferences in bases flanking the core sequence (Fisher et al., 1993; Solomon et al., 1993). In addition to analyzing c-Myc/Max and Max/Max dimers, the COS-7 system will also enable studies of DNA-binding parameters of in vivo derived complexes containing Mad family members (Bousset et al., 1995; Cerni et al., 1995).

D. Myc as a Transcription Factor The identification of c-MycIMax DNA-binding sequences made possible the design of Myc responsive reporter gene constructs and evaluation of the putative transactivating function of Myc. Using synthetic reporter plasmids containing the Myc/Max binding site it was demonstrated both in yeast and in mammalian cells that Myc/Max complexes activate transcription in a sequence-specific manner, whereas Max homodimers repress transcription (Amati et al., 1992; Kretzner, et al., 1992; Amin et al., 1993; Gu et al., 1993). These reporter constructs were further used to characterize transcriptionally relevant regions in c-Myc. As expected from the DNA-binding studies, the basic region and the HLHZip domains are critical for Myc’s ability to induce transcription (Kretzner et al., 1992; Amin et al., 1993; Gu et al., 1993). The other functionally important region in c-Myc is the N-terminal domain of roughly 150 amino acids. Using GAL4-Myc fusion proteins it

I24

Marie Henriksson and Bernhard Luscher

was deduced that the N-termini of c-Myc and of v-Myc have the capacity to activate gene transcription (Kato et al., 1990; Min and Taparowsky, 1992). The analysis of c-Myc deletion mutants covering the first 200 amino acids of Myc supports the concept that the N-terminal domain is important for transactivation (Amati et al., 1992; Kretzner et al., 1992; Amin etal., 1993; Gu et al., 1993). However, a more detailed mapping has been hampered since the data obtained using either GAL4-Myc or full-length c-Myc were only partially compatible (see also Sections IV,B, and VII,E). It was reported that a Myc fragment can bind to the C/EBP sequences within the EFII enhancer element of the Rous sarcoma virus LTR (Hann et al., 1994). Interestingly, Myc-1 and Myc-2 (or p67 and p64 Myc) seem to differ in their ability to transactivate reporters containing this alternative binding site. At present the relevance of these findings in regard to the function of Myc in growth control is not understood. I . MAX IS ESSENTIAI FOR C-MYC-DEPENDENT TRANSACTIVATION

The mutational analysis of c-Myc had indicated that the C-terminal HLHZip region is essential for transactivation. Because this is the domain which interacts with Max, and c-Myc requires heterodimerization with Max for DNA binding, these findings are all consistent. Overexpression of Max has been shown to efficiently inhibit transactivation from promoter constructs with c-Myc/Max consensus sequences (Amati et al., 1992; Kretzner et al.. 1992; Amin et al., 1993; Gu et al., 1993). This is explained by the lack of a transactivating domain in Max and by the ability of Max to form homodimers which compete with activating c-Myc/Max complexes. Indeed, if Max is fused to a heterologous TAD it now becomes an activator (Amin et al., 1993). Recent findings made in the pheochromocytoma cell line PC12 support the essential role Max performs in c-Myc transactivation. It was demonstrated that PC12 cells do not express functional Max due to aberrant processing of the max mRNA (Hopewell and Ziff, 1995). The resulting transcript encodes a protein incapable of homo- or heterodimerization and which therefore cannot repress transcription from an E-box element. It was found that c-Myc is unable to transactivate reporter genes in PC12 cells, whereas cotransfection of Max led to a dose-dependent repression in both the presence or the absence of exogenous c-Myc (Bousset et al., 1994; Hopewell and Ziff, 1995). In contrast to these findings, another group reported stimulation of a reporter gene construct by c-Myc in PC12 cells (Ribon et al., 1994). Since these authors were also unable to detect Max in their PC12 cells, the discrepancy between the results with c-Myc is not understood at present.

125

Myc Proteins

2. EFFECTS O F MAD FAMILY MEMBERS ON C-MyC-ACTIVATED TRANSCRIPTION Several lines of research have suggested that the Mad family proteins may antagonize c-Myc function. The first report of Mad showed that it is an efficient inhibitor of c-Myc-dependent transactivation in NlH3T3 cells (Ayer et al., 1993). This led to the suggestion that Mad may compete with c-Myc for Max binding thereby inhibiting transactivation. More recently the N-terminal domains of Mad family proteins have been shown to interact with mSin3A and mSin3B, the murine homologues of the yeast transcriptional repressor Sin3 (Ayer et af., 1995; Hurlin et a/., 1995; Schreiber-Agus et al., 1995; see Fig. 4). This finding suggested that Mad does not simply compete with Myc (which it may also do) but that it additionally recruits a repressor protein to c-MycJMax binding sites. As shown for Mad, Mad3 and Mad4 also repress CACGTG-mediated transcription efficiently (Hurlin et al., 1995a). Although no transactivation data for Mxil are available, all the analyses performed to date point to a similar mode of action of M x i l as proposed for the other Mad proteins (Schreiber-Agus et al., 1995). The identification and characterization of mSin3A and mSin3B adds to the complex pattern of interacting factors that appear to control the activity of the Myc network proteins. Yeast Sin3 is a nuclear protein consisting of 1538 amino acids with four putative paired amphipathic helix (PAH) domains (analogous to the HLH motif ), which are believed to mediate proteinprotein interactions (Wang and Stillman, 1993 and references therein). The murine sin3A and sin3B cDNAs encode open reading frames of 1219 and positive regulation of cell growth

I

negative regulation

II

of cell growth

I

Mad3 Mad4 i

ij

CACGTG -GTGCAC Fig. 4 The Myc network proteins and associated factors are shown. In vivo interactions between the different proteins are indicated by arrows.

126

Marie Henriksson a n d Bernhard Luscher

954 amino acids, respectively, with all four PAH domains conserved (Ayer et al., 1995). The HR1 domains of Mad family proteins mediate interaction with the PAH2 motif of the mSin3 proteins. It was demonstrated that Mad proteins bearing mutations in the amino-terminal region neither could bind mSin3 proteins in vitro nor repress CACGTG-mediated transcription in uivo, suggesting that the mSin3-Mad interaction is necessary for transcriptional repression by Mad (Ayer e t al., 1995). Since yeast Sin3 does not bind to DNA on its own (Wang and Stillman, 1990) and since it was previously shown that transcriptional repression by Mad is dependent on its ability to form DNA-binding heterodimers with Max, it has been suggested that Mad/Max heterodimers might recruit mSin3 proteins as corepressors to the DNA (Ayer et al., 1995). Supporting this hypothesis are the findings that mSin3A or B, Max, and Mad can form a ternary complex with DNA in vitro, as well as data showing a ternary complex between Max, Mxil, and the mSin3B PAH2-containing region in vivo using expression constructs (Ayer et al., 1995; Schreiber-Agus et al., 1995). Furthermore, the observation was made that Mxil-WR, which lacks HR1, had a reduced repressive potential on myclras cotransformation arguing for the functional relevance of the mSin3 interaction domain (Schreiber-Agus et al., 1995; see Section VI1,F). These findings further indicate that the Myc antagonizing function of Mad proteins is not explained by simple competition. The recruitment of transcriptional repressors to the DNA by sequence-specific DNA-binding proteins may be a common mechanism by which gene expression is downregulated. Once in contact with a promoter, Sin3 proteins may exert their repressive effects by modifying the activity of other transcriptional regulators or by maintaining the surrounding chromatin in a repressed state (Wang and Stillman, 1993). Whereas the repression of c-Myc-responsive reporter genes by Mad appears straightforward in NIH3T3 cells, n o repression was observed in a number of other cell lines including rat embryo fibroblasts (REFS) (Cerni et al., 1995; C. Dang, M. Eilers, personal communications). It is possible that the mSin3 levels are an important determinant of Mad’s ability to repress. With the availability of mSin3 expression clones it will now be possible to test whether these proteins are limiting.

E. Proteins Interacting with c-Myc An increasing number of proteins have been suggested to interact with c-Myc such as the retinoblastoma susceptibility protein Rb, the Rb-like p107 protein, the transcriptional regulators Yin-Yang-1 (YY-1) and AP-2, the TATA-binding protein (TBP), TFII-I as well as a-tubulin (Rustgi et al., 1991; Hateboer et af., 1993; Roy et al., 1993; Shrivastava et al., 1993; Beijersbergen et al., 1994; Gu et al., 1994; Alexandrova et al., 1995;

Myc Proteins

127

Gaubatz et al., 1995). An in vivo interaction has been shown for TBP, p107, AP-2 and a-tubulin (Beijersbergen et al., 1994; Gu et al., 1994; Maheswaran et ul., 1994; Alexandrova et ul., 1995; Gaubatz et ul., 1995) and recently for YY-1 (M. Austen and B. Liischer, unpublished observation) (see Fig. 4). Microinjection of bacterially expressed c-Myc protein could abrogate cell cycle arrest induced by Rb (Goodrich and Lee, 1992) suggesting some functional link between the two proteins. Furthermore, the amino-terminal region of Myc has been implicated in complex formation with the Rb protein (Rustgi et al., 1991). These findings suggested a model in which a tumor suppressor regulates Myc activity through direct binding; however, an in vivo interaction of c-Myc with Rb has not been observed (Beijersbergen et al., 1994). The use of a mammalian two hybrid system has revealed conflicting data. In one report no interaction between Rb and Myc was detected, supporting the previously described findings (Hoang et al., 1995; see also below). In a more detailed analysis a functional interaction of Rb with Myc was observed, resulting in a stimulation of Myc-dependent transactivation in some, but not all, cell lines (Adnane and Robbins, 1995). Mapping of the interaction domains indicated that the TAD of GAL4-Myc and the pocket region B of Rb were important. These latter findings revive the concept of a functional link between these important cell growth regulators. Unlike the findings with Rb discussed above, a specific complex between Myc and p107 has been demonstrated in vivo (Beijersbergen et al., 1994; Gu et al., 1994). This interaction occurs through the pocket region of p107 and the N-terminus of c-Myc. Similarly, adenovirus E1A as well as SV40 T antigen bind to the pocket region of pRb and p107, thereby overriding their growth inhibitory effects. Therefore it has been speculated that Myc exerts a similar function. Furthermore, it has been shown that the binding of p107 to Myc causes a significant inhibition of Myc-mediated transcriptional activation (Beijersbergen et al., 1994; Gu et al., 1994; Hoang et al., 1995). This is in contrast to the finding with Rb (Adnane and Robbins, 1995), a discrepancy which is surprising and requires further analysis. Both wild-type and mutant forms of Myc from Burkitt Lymphoma (BL) cells, which contain point mutations in the TAD, were shown to bind p107 with similar efficency. However, transactivation by the Myc mutants was no longer suppressed by p107 (Gu et al., 1994; Hoang et al., 1995). This finding is controversial since similar or identical Myc mutants were repressed equally well compared to wild-type c-Myc in a second study (R. Bernards, personal communication). In view of these conflicting data the molecular consequence of myc exon 2 mutations frequently found in BLs remains illusive. However, one explanation has been offered by the correlation between the resistance of c-Myc mutants to p107 suppression and the lack of cyclin A-dependent phosphorylation of c-Myc (Hoang et al., 1995). p107 has been suggested to tether Myc to a cyclin A/CDK complex which can phosphory-

I28

Marie Henriksson and Bernhard Luscher

late wild-type c-Myc but not the mutant Myc proteins derived from BL. It is possible that mutations even relatively distant from the phosphorylation sites at Thr-58 and Ser-62 alter the structure of the Myc N-terminus resulting in inhibition of phosphorylation through the associated CDK and thereby affecting the growth promoting properties of c-Myc (see Section VII). Taken together these data are appealing because they suggest a direct regulation of Myc activity by a putative tumor suppressor. Furthermore, they implicate a means by which Myc escapes this regulation, i.e., by acquiring mutations that inhibit plO7-mediated growth control. This may provide one step toward the full activation of the oncogenic potential of c-Myc in Burkitt lymphoma. YY-1 regulates the transcription of many genes and can, depending on the context, act as a transcriptional repressor, activator, or initiator (see Shrivastava and Calame, 1994). Using the yeast two hybrid screen for proteins that interact with YY-1, a c-myc cDNA was isolated. It was shown that the C-terminal region comprising residues 250-439 of c-Myc is involved in the association with YY- 1 and that this interaction prevents dimerization with Max (Shrivastava et al., 1993). Furthermore, the binding of Myc to YY-1 inhibited the repressor as well as the activator functions of YY-1, suggesting an additional mode of action of Myc, independent of its direct binding to DNA. Since YY-1 is a much more abundant protein than c-Myc this regulation may only apply under conditions where the c-Myc expression is elevated, for instance in certain tumors. Interaction of c-Myc with yet another transcription factor has been reported recently. A direct association of AP-2 with the bHLHZip domain of c-Myc was observed (Gaubatz et al., 1995). This interaction does not interfere with the binding of Max to c-Myc but prevents DNA binding and concomitantly Myc-specific transactivation. Together with the identification of overlapping c-Myc and AP-2 response elements in several promoters (see Section IV,B), the interplay of these two transcription factors therefore offers a number of regulatory possibilities. Transcriptional activation domains are thought to attract other factors required for facilitated transcriptional activation o r repression. In line with this is the finding that several viral transactivators, including adenovirus E l A, herpes simplex VP16, and Epstein-Barr virus Zta, can interact directly with TBP, the TATA-binding component of the basal transcription factor TFIID (Lieberman and Berk, 1990; Stringer et al., 1990; Lee et af., 1991). The observation that the N-terminus of c-Myc also physically interacts with TBP both in vitro and in vivo supports the notion that Myc is a transcription factor and indicates that Myc is able to conduct its transactivating function directly through the basal transcriptional machinery (Hateboer et al., 1993; Maheswaran et al., 1994; Hoang et al., 1995). Myc has also been shown to interact with another component of the

Myc Proteins

I29

general transcriptional machinery, namely, the initiator-binding protein TFII-I (Roy et al., 1993). Both the HLH and the Zip of Myc are important for the interaction and Myc thereby inhibits TFII-I-dependent transcription from the initiator element. These data suggest that Myc might exert different effects on different promoters depending on the presence o r absence of a n initiator element. In its presence Myc would repress, whereas in the absence of an initiator Myc could activate transcription through an E-box. However, these in vitro data require further substantiation. In some rare instances the subcellular localization of Myc has been reported as cytoplasmic (see Section V,A). Since translocation from the cytoplasm into the nucleus of certain proteins requires microtubules, it was tested if Myc can interact with such structures. A region within the TAD of c-Myc interacted in vivo with a-tubulin and microtubules suggesting that these could be involved in regulating Myc function (Alexandrova et al., 1995). One possibility would be to effect the nuclear-cytoplasmic distribution of c-Myc. It will be important to determine the regulation of the c-Myc/a-tubulin interaction and to analyze the properties of the microtubule-associated population of Myc proteins. In a novel bacterial expression screen a high mobility group (HMG)-box protein, SSRP1, was identified by means of its association with the c-Myc bHLHZip domain (Bunker and Kingston, 1995). Although these authors could not establish interaction in any other system tested than the original bacterial screen, the C-terminal portion of SSRPl was shown to inhibit c-Myc-mediated transactivation in transient transfection experiments. Using an interaction trap in yeast a novel Myc-interacting protein called 99 has been identified (D. Sakamuro and G. Prendergast, personal communication). This protein interacts with the amino terminus of Myc and suppresses Myc-dependent cell transformation in REFS. The data reviewed in this section draw a complex picture of many different proteins capable of binding to c-Myc. Further detailed characterization of the biological relevance of these Myc partners as well as hitherto unidentified ones will help to shed light on the nature of Myc regulation and function.

IV. Myc TARGET GENES A. Indirect a n d Direct Target G e n e s A considerable number of reports have suggested that c-Myc is involved in the regulation of gene transcription. However, the initial reports did not provide evidence for a direct role of c-Myc in gene regulation. It was found

130

Marie Henriksson and Bernhard Liischer

that c-Myc could activate the heat-shock promoter and repress the metallothionein promoter (Kingston et al., 1984; Kaddurah-Daouk et al., 1987). The adenovirus E4 promoter is also c-Myc inducible and the element involved appears to overlap the region conferring E l A responsiveness (Onclercq et al., 1988). Because this element does not contain an E-box the c-Myc effect is likely to be indirect. Furthermore, the mrl (encoding plasminogen activator inhibitor 1, PAI-1) and mr2 genes were shown to be positively regulated by c-Myc (Prendergast and Cole, 1989; Prendergast et al., 1990). A number of other studies described repression of genes upon expression of high levels of c-Myc or N-Myc. These include the genes encoding M H C class I antigens, HLA class I antigens, lymphocyte function-associated antigen 1, neural cell adhesion molecule, and collagenase (Bernards et al., 1986; Versteeg et al., 1988; Akeson and Bernards, 1990; lnghirami et al., 1990; Yang et al., 1991, 1993; Peltenburg et al., 1994). Currently it is unclear how Myc influences the expression of most of these genes. In the case of M H C class I antigens a role for NFKB transcription factors has been demonstrated, providing evidence for an indirect action of N-Myc (van’t Veer et al., 1993). The repression of transcription of some of the previously mentioned genes could be of importance in tumor progression, i.e., in evading an immune response o r in altering cell-cell o r cell-substratum communication. Mechanistically, repression by c-Myc is not understood. It has been suggested that c-Myc may negatively regulate gene transcription through the initiator regions of promoters, such as the adenovirus major late and the cyclin D 1 promoters (Li et al., 1994; Philipp et al., 1994; see also below). However, the data published are only partially consistent. Whereas Myc Box I1 and the interaction with Max are important for repression of the adenovirus major late promoter (Li et al., 1994), the repression of the cyclin D1 promoter requires aa 92-106 in Myc and is independent of Max (Philipp et al., 1994). In addition, it is unclear if Myc can act directly on the initiator, for instance by interacting with an initiator binding protein, or if the effects are indirect. Clearly more detailed analysis will be required to firmly establish a role of c-Myc in the regulation of initiator elements. The ability of c-Myc to autorepress its own promoter has been well documented. This was first observed in the analysis of the expression of the myc genes in Burkitt lymphomas. Whereas the translocated allele is expressed at high levels in these tumors, the unaltered allele is silent (for review see Spencer and Groudine, 1991). Furthermore, the introduction of Myc into cells of various origins repressed endogenous myc genes (Grignani et al., 1990; Penn et al., 1990a,b). Myc family members also repress each other reciprocally (see DePinho et al., 1991). Because we now know that c-Myc not only drives cell growth but also can lead to apoptosis, such a feedback

Myc Proteins

131

regulation could be relevant to ensure appropriate Myc expression. At the molecular level a number of suggestions have been presented including autorepression through YY-1 response elements (Shrivastava et al., 1993) o r through effects on initiator sequences at the P2 promoter (L. Kretzner, personal communication; Krumm et al., 1993). Interestingly, this latter effect requires Myc Box 11, like the repression of the adenovirus major late promoter (L. Kretzner, personal communication). Furthermore, it has been suggested that c-myc autosuppression occurs through an indirect mechanism (Grignani et al., 1990; Buckle and Mechali, 1995). Together these data indicate that c-myc autoregulation appears to be the result of multiple regulatory events. The protein products of the genes described above do not explain the dominant and essential role Myc plays in cell growth control. Furthermore, little evidence for their direct regulation has been obtained. A number of criteria can be established which should be fulfilled to make a gene a good candidate for being a direct Myc target. The simplest prediction is that expression of a direct target gene should correlate with the expression pattern of Myc; that is, it should respond to the differential expression of c-Myc observed during the transition from GO to G1, from cycling to quiescent cells, or when cells proceed to differentiate. The gene should be expressed at a constant rate during the cell cycle because c-Myc is expressed constitutively and it should be elevated in tumors with overexpressed c-Myc. Mycregulated genes, at least those relevant for Myc’s effect on cell growth control and transformation, should be sensitive to mutations in c-Myc known to compromise function. Furthermore, a promoter element should be present in the putative target gene which can bind c-MyclMax complexes and which is relevant for the appropriate expression of the gene, specifically this element has to mediate the c-Myc effect. Finally, this element should be occupied in intact cells at the appropriate times. This may at least in part be too simplistic. It is unlikely that the transcription of any gene will merely follow Myc expression in vivo. c-Myc may be regulated post-translationally, for instance, by phosphorylation altering its specific transactivation capacity. In addition, because promoters in general are regulated by multiple elements it is likely that the activity of c-Myc is embedded in the activities of a number of additional transcription factors; the combined action of these will determine the activity of a given promoter. Several transcription factors have been described which in specific circumstances provide significant promoter activation, including the SRF/TCF complex binding to the serum response element or CREB on the CAMP response element (see Lalli and Sassone-Corsi, 1994; Janknecht et al., 1995). These proteins are specifically targeted by signal transduction pathways resulting in rapid and transient induction of gene transcription. Because

132

Marie Henriksson a n d Bernhard Luscher

little evidence for signal transduction onto c-Myc has been obtained, and its transactivation capacity is rather modest, it seems likely that c-Myc will perform its role in gene regulation in cooperation with other proteins. However, at present no such factors have been identified. Different approaches have been taken to identify direct Myc-regulated genes. The experimental designs relied on differential screening of mRNA obtained from cells expressing either high or low levels of c-Myc. Myc responsive genes include the a-prothymosin gene, the ornithine decarboxyfuse ( O D C )gene, the p.53 tumor suppressor gene, the ECA39 gene, the cad gene, and several cyclin genes (Eilers et af., 1991; Benvenisty et af., 1992; Bello-Fernandez et al., 1993; Jansen-Durr et al., 1993; Reisman et al., 1993; Wagner et af., 1993a; Daksis et al., 1994; Philipp et al., 1994; Miltenberger et al., 1995). All these genes fulfill at least part of the previously mentioned criteria for being directly regulated by Myc, and will be discussed in more detail below.

B. a-Protkymosin The a-prothymosin gene encodes an acidic nuclear protein of unknown function. Its expression appears to correlate with cell growth and the protein has been suggested to be essential in myeloid cells (Conteas et al., 1990; Bustelo et af., 1991; Manrow et af., 1991; Sburlati et al., 1991; Smith et af., 1993). The a-prothymosin gene was identified as Myc inducible in quiescent fibroblasts using chimeras of c-Myc and the ligand-binding domain of the human estrogen receptor (c-MycER). This protein can be activated upon addition of hormone as demonstrated in transformation assays (Eilers et al., 1989,1991). Although this gene can be induced by Myc in quiescent cells, it is not induced by activating c-MycER in growing cells. It appears that in such cells a-prothymosin has a high basal activity which cannot be further stimulated by Myc. A more detailed analysis of the regulation of the a-prothymosin gene by c-Myc indicated that the responsive element is localized in the first intron of the gene and contains a 5'-CACGTG consensus sequence (Gaubatz et al., 1994). Interestingly, the expression of a-prothymosin mRNA correlates with that of c-myc in human colorectal cancer and during the differentiation of HL-60 promyelocytic cells (Dosil et al., 1993; Mori et af., 1993; Smith et al., 1993). The location of the response element in the a-prothymosin gene is reminiscent of the other genes suggested to be regulated by c-Myc. The genes coding for ODC, pS3, CAD, and ECA39 all possess Myc/Max response elements 3' relative to the start of transcription (see below). Currently it is unclear if the positioning of the Myc/Max response element is relevant for the ability of Myc to regulate these genes. However, two recent findings are of consider-

Myc Proteins

133

able interest. The first is that significant differences can be observed when the transactivation activity of mutants in the TAD of c-Myc are compared on a synthetic reporter gene containing several MycIMax binding sites upstream of a minimal promoter, with a reporter gene construct containing the a-prothymosin promoter including exon 1, intron 1 with the Myc responsive element, and part of exon 2. Most important, mutations in Myc Box I1 can no longer activate transcription from the a-prothymosin construct, whereas they are still able to d o so from the mini reporter gene (M. Eilers, personal communication). This indicates a correlation between Myc’s ability to transactivate the a-prothymosin promoter and to transform, because Myc Box I1 is also essential for Myc-dependent transformation. The establishment of a relationship between transactivation and transformation in relation to Myc N-terminal sequences has not been straightforward using synthetic reporter gene constructs, bringing into question their biological significance (see also Section VII,E). Second, it appears that USF, which can bind to a MycIMax consensus sequence, cannot transactivate from the site in the first intron of the a-prothymosin gene, whereas it can if the same site is placed close to a minimal promoter, suggesting specificity due to the location of the response element (M. Eilers, personal communication). In comparable experiments using the ODC gene no difference between the ability of c-Myc and USF to transactivate has been observed (C. Dang, personal communication). However, the Myc-responsive element in the ODC gene is located closer to the promoter than in the a-prothymosin gene, which may provide an explanation for the different USF effects observed. These interesting findings suggest that the position of the c-Myc response element relative to the basal promoter sequences may provide some specificity although at present n o general conclusion can be drawn. In addition these results indicate a difference in how c-Myc and USF enhance transcription. The ability of Myc to transactivate from a distal enhancer element may be a consequence of its ability to interact with the coactivator CBP (M. Austen, unpublished observation). It has been noted that the Myc responsive element in the a-prothymosin, as well as in several other genes, is located close to an AP-2 site. AP-2 appears to compete with c-MycIMax for binding and this competition inhibits Myc transactivation (Gaubatz et af., 1995). This may provide a novel mechanism to regulate Myc-specific gene transcription.

C. Ornithine Decarboxylase O D C is the rate-limiting enzyme for polyamine biosynthesis and essential for progression into S phase. Analysis of its expression pattern has shown

I34

Marie Henriksson and Bernhard Liischer

that the gene is induced in mid-G1 in response to a wide variety of stimuli including growth factors, steroid hormones, and substances inducing CAMP production (Abrahamsen et al., 1990; Heby and Persson, 1990; Hibshoosh et al., 1991). Overexpression of ODC in NIH3T3 fibroblasts results in transformation (Auvinen et a/., 1992; Moshier et al., 1993), whereas the expression of an antisense construct not only can reduce cell growth but also prevents transformation by V-SYC (Auvinen et al., 1992). The analysis of the O D C promoter revealed several potential c-Myc/Max binding sites, the two positioned in the first intron are conserved in mammals (see Bello-Fernandez et al., 1993). These sites can bind both Max/Max homo- and c-Myc/Max heterodimers. A reporter construct encompassing upstream promoter regions, exon 1, intron 1, and part of exon 2, was shown to be responsive to c-Myc in an E-box-dependent manner. Mutational analysis of c-Myc indicated that both the TAD and the bHLH regions were relevant for transactivation. Unexpectedly, deletion of the Zip domain resulted in a mutant Myc protein with enhanced transactivational potential. Because this protein cannot bind to Max and consequently will not bind DNA, this finding is difficult to reconcile with current models of c-Myc transactivation. Using the c-MycER system it was shown that the endogenous O D C gene can be turned on in serum-starved cells upon addition of hormone (Wagner et al., 1993a). O D C transcription is activated in the presence of protein synthesis inhibitors indicating that this gene is a direct target for c-Myc.

D. p53 The tumor suppressor protein p53 plays an important role in the cellular response to DNA damage (Vogelstein and Kinder, 1992; Greenblatt et al., 1994). p53 can, upon activation, inhibit cell cycle progression from G1 to S, and thereby inhibit replication (for review see Selivanova and Wiman, 1995). pS3 acts at least in part through the transcriptional activation of the p21 gene which encodes a potent inhibitor of several cyclin-dependent kinases (El-Deiry et d.,1993). A c-MycIMax consensus binding sequence was identified downstream of the start of transcription in the murine p53 gene. This element has previously been shown to be important for maximal promoter activity and responsiveness to c-Myc (Ronen et al., 1991; Reisman et al., 1993). This site is only partially conserved in human p53 and replaced by a sequence with lower affinity for c-MycIMax. Nevertheless, the human p53 promoter can be activated by c-Myc (Roy et al., 1994). A priori it does not seem very obvious why c-Myc should activate the transcription of p53 since c-Myc is Strongly associated with cell proliferation whereas p53 inhib-

Myc Proteins

135

its growth, Quiescent fibroblasts will enter S phase upon activation of cMycER and concomitantly p53, at least in part due to increased transcription, and p21 are induced (Hermeking and Eick, 1994; Wagner, et al., 1994). In this respect it is interesting to note that a correlation between the expression of c-Myc and p53 has been observed in a number of tumor cell lines, and that repression of c-Myc with antisense RNA results in a decrease in p53 (Roy et al., 1994). Because c-Myc overexpression can have severe effects on cell fate, including inhibition of differentiation and promotion of tumor development, the activation of p53 could represent a safeguard mechanism. Pathologically elevated c-Myc levels could lead to increased transcription of p53 and concomitantly to apoptosis (see also Section VI).

E. ECA39 In a differential screen using mRNA from a brain tumor induced by overexpression of c-myc and from normal brain tissue, a novel gene called ECA39 was identified which appears to be Myc regulated (Benvenisty et al., 1992). Similar to the previously described O D C and a-prothymosin genes, ECA39 contains a c-MycIMax consensus sequence located downstream of the transcriptional start site in the 5’ untranslated region of the gene. Mutational analysis indicates that this element is functional in transient transfection assays. At present the function of the ECA39 gene product is unclear.

F. cad Cad encodes a multifunctional enzyme required for the de novo synthesis of pyrimidines. The expression of cad is induced late in G 1 near the transition into S phase and at least part of this induction appears to be transcriptional. The response element relevant for this cell-cycle-specific expression contains binding sites for E2F transcription factors and a c-MycIMax E-box sequence. It has been suggested that Myc/Max complexes contribute to the activity of this E-box which is essential for the induction of the cad gene at the GI-S transition (Miltenberger et al., 1995).

G. G e n e s Encoding Cell Cycle Regulators The potent role c-Myc has in driving the cell cycle is best exemplified in the c-MycER system. c-MycER-expressing fibroblasts made quiescent in the absence of serum can be stimulated to reenter the cell cycle and proceed

I36

Marie Henriksson and Bernhard Luscher

beyond the restriction point by simply activating latent c-MycER. It was therefore of interest to test if the expression of known cell cycle regulators can be modulated by c-Myc. Cyclin A and cyclin E transcription is enhanced in response to c-Myc and this is paralleled by an increase in E2F activity (Jansen-Durr et al., 1993). It appears that the expression of cyclin A is downstream of the activation of cyclin E and not a consequence of direct activation by c-Myc (L. Penn, M. Eilers personal communications). However, these two cyclins are required late in G1 and in S and are unlikely to explain the effects of c-Myc on cell cycle progression early in G1. The genes encoding CDKl (p34cdc2) and CDK2 have also been reported to be c-Myc inducible (Born et al., 1994; Kim et al., 1994). In the study mentioned previously, cyclin D1, in contrast to cyclins A and E, was found to be repressed by both constitutive and inducible c-Myc in Balb/c-3T3 cells (Jansen-Durr et al., 1993). This repression was suggested to be mediated through the initiator element of the cyclin D 1 gene and in the case of c-MycER occurred even in the absence of hormone (Philipp et a/., 1994). In contrast, an induction of cyclin D1 expression in response to c-MycER activation was observed in Rat1 cells (Daksis et al., 1994). These authors demonstrated that the induction is rapid and independent of protein synthesis, arguing for a direct role of c-Myc in the activation process. Indeed, a c-Myc/Max response element has been identified relevant for the observed activation. Induction of cyclin D1 by c-Myc has also been observed in NlH3T3 cells and was suggested to be indirect through activation of the initiation factor 4E (Rosenwald et al., 1993a,b). In yet another study, n o effect of c-Myc on cyclin D1 expression was detected (Hanson et al., 1994). While these findings appear confusing, it is evident that cyclin D1 is strongly linked to cell growth (Hunter and Pines, 1994; Sherr, 1994). Cyclin D1 is required in certain cell systems to traverse G1, and overexpression is linked to a growing number of tumors (cited in Daksis et al., 1994). Furthermore, cyclin D1 has been shown to cooperate with activated Ras in the transformation of REFS, supporting an important role of cyclin D 1 in growth control (Lovec et al., 1994b). Some recent findings appear to shed light on the role of Myc in the regulation of cyclin D1. The induction of cyclin D1 by c-MycER using P-estradiol (Daksis et al., 1994) appears to be due to the activation of a cryptic TAD in the hormone-binding domain. This can be prevented by using the estrogen agonist hydroxytamoxifen (Solomon et al., 1995). It also seems that the Myc effects on cyclin D1 are cell-type dependent (Marhin et al., 1995; Solomon et al., 1995; Steiner et al., 1995). One possible explanation for these differences could be the status of the Rb protein in these cells. By comparing the effect of c-Myc in mouse embryo fibroblasts from Rbdeficient animals and normal litter mates it became evident that repression

Myc Proteins

137

of cyclin D1 by Myc occurred only in the former and not in the latter (Marhin et al., 1995). The analysis of cyclin-dependent kinases has now revealed that the induction of c-MycER leads to an activation of both cyclin D1- and cyclin E-associated kinase activity with concomitant Rb hyperphosphorylation (Steiner et al., 1995). This occurs in the absence of any increase in either cyclin o r kinase expression, suggesting that cyclin-dependent kinase inhibitors (CKI) might be regulated in response to the induction of c-MycER. Indeed, a rapid decline of p27 is observed upon addition of hydroxytamoxifen (Steiner et al., 1995). Recently, it has been shown that the abundance of p27 in cells is regulated mainly by modulating protein stability (Pagano et al., 1995).Since p27 degradation is mediated by the ubiquitin-proteasome pathway, it is conceivable that c-Myc may modulate p27 half-life by regulating genes involved in the ubiquitination of p27 or in proteasome function. An even more complex relationship between cyclin D1 and Myc has been suggested by the analysis of cells expressing CSF-1 receptor mutants, which are unable to induce c-myc transcription in response to CSF-1. These cells d o not induce cyclin D1, fail to proliferate, and arrest in the early G1 phase of the cell cycle (Roussel et al., 1990, 1991, 1994), but can be resensitized to mitogenic signals by ectopic expression of either c-Myc or cyclin D1 (Roussel et al., 1991, 1995). In cells expressing c-Myc, induction of cyclin D1 by CSF-1 was restored and vice versa, suggesting that the expression of c-Myc and cyclin D1 is interdependent. However these events are still poorly understood at the molecular level.

V. THE ROLE OF Myc NETWORK PROTEINS IN THE CELL CYCLE AND DURING DIFFERENTIATION

A. Myc in Cell Proliferation Myc is continuously expressed during the cell cycle but its levels change rapidly in response to agents which stimulate or repress proliferation. In nonproliferating o r growth-arrested (i.e., GO) cells myc mRNA and protein are essentially undetectable. Following serum stimulation there is a rapid increase in Myc levels within several hours after induction, followed by a relatively slow decline commencing well before the onset of the first S phase (Kelly et al., 1983; Campisi et al., 1984; Greenberg and Ziff, 1984; see Fig. 5). The rapid and transient induction of myc during the GO/G1 transition, which does not require new protein synthesis, is characteristic of the “early response” genes, such as c-jun and c-fos. In contrast to many genes of this

138

Marie Henriksson and Bernhard Luscher

Differentiation

I

G O d G1 predominant Max/Max

- Cycling cells

Differentiating cells

protein complexes: Myc/Max

Myc/Max Max/Max

Myc/Max Mad/Max

Mad/Max M x i l /Max

Fig. 5 Expression pattern of the proteins in the Myc network in hematopoietic cells. Predorninant protein complexes at different stages of the cell cycle as well as during differentiation are indicated.

class, myc levels do not return to background following entry into the cell cycle, but are maintained at a constant level throughout all phases of the cell cycle in continuously proliferating cells (Hann et al., 1985; Rabbits et al., 1985; Thompson et al., 1985). The only cell cycle-dependent variation in c-Myc detected to date is hyperphosphorylation during mitosis (Liischer and Eisenman, 1992), an event that accompanies nuclear envelope breakdown and redistribution of Myc in the cytoplasm. Both c-Fos and c-Myc are important for progression into S phase, and several lines of evidence have indicated that the expression of the corresponding genes is regulated by different pathways. For fos, signaling by Ras appears critical (Janknecht et al., 1995). Now new information identifies the tyrosine kinase Src as an upstream activator of c-myc, but not c-fos, transcription (Barone and Courtneidge, 1995). These findings demonstrate separate, molecularly defined pathways leading to the activation of c-myc and c-fos. Cells constitutively expressing high levels of Myc have reduced growth factor requirements, increased growth rate, spend less time in G1, and can in some cases circumvent growth arrest (Palmieri et al., 1983; Armelin et al., 1984; Sorrentino et al., 1986; Stern et al., 1986; Kohl and Ruley, 1987; Karn et al., 1989). In support of myc being an early response gene, deletion of one copy of c-myc by homologous recombination in a nontransformed rat

Myc Proteins

I39

fibroblast line resulted in reduced c-myc expression, slower growth rate, and a delayed entry into S phase upon serum stimulation (Shichiri et al., 1993). Furthermore, the inhibition of Myc expression in mitogenically activated T cells by antisense oligonucleotides prevents entry into S phase (Heikkila et al., 1987). In addition, using the c-MycER system, it has been shown that activation of c-Myc is sufficient to stimulate DNA synthesis of quiescent cells (Eilers et al., 1991). The data previously summarized demonstrate that c-Myc is essential for progression through the G 1 phase of the cell cycle. However, the constitutive expression of c-Myc in growing cells and its rapid decline upon withdrawal of growth factors throughout the cell cycle (Waters et al., 1991) suggest functional relevance also in the S and G2 phases of the cell cycle. Myc proteins have been found to be localized in the cell nucleus in most instances studied (see DePinho et al., 1991). One exception exists during Xenopus oogenesis where c-Myc is accumulated in the cy' 71 ..d at least part of it translocates into the nucleus upon fertilization (Taylor et al., 1986; Gusse et al., 1989). Recent evidence indicates that of the two predominant Myc proteins only the p64 translocates into the nucleus, whereas p61 remains cytoplasmic (Lemaitre et al., 1995). Surprisingly, no interaction of c-Myc with Max and concomitantly no DNA binding was observed. This seems to be the result of an activity present in extracts of eggs and early embryos capable of disrupting Myc/Max complexes (Lemaitre et al., 1995). An altered subcellular distribution of c-Myc has also been described in human ML-1 myeloid leukemia cells. Upon TPA-induced differentiation c-Myc was found predominantly in the cytoplasm (Craig et al., 1993). Similarly, N-Myc redistribution to the cytoplasmic compartment was observed upon differentiation of specific classes of neurons (Wakamatsu et al., 1993). These findings suggest an alternative mechanism to negatively regulate Myc function during differentiation. Growing cells normally have low levels of p67Myc compared with p64Myc. However, as cells approach high density in culture, there is a sustained 5- to 10-fold increase in the synthesis of p67Myc to levels greater than or equal to the levels of p64Myc synthesis (Hann et al., 1992). The functional relevance of this switch is unclear.

B. Expression Pattern of the myc Family Genes During murine embryogenesis c-myc is expressed in all tissues but the levels fluctuate over various developmental stages (Schmid et al., 1989). In the adult mouse c-myc is expressed in some tissues like thymus, spleen, kidney, liver, and intestine (Zimmerman et al., 1986; Semsei et al., 1989). A lower level of expression is found in adrenal gland, brain, lung, and heart

140

Marie Henriksson and Bernhard Luscher

(Zimmerman et al., 1986). Similar findings have been reported in Xenopus and chicken tissues (Gonda et al., 1982; King et al., 1986). c-myc is expressed in proliferating cells during all stages of B and T cell differentiation (Zimmerman et al., 1986; DePinho et al., 1991). The analyses of the developing human placenta (Pfeifer-Ohlsson et al., 1984), regenerating murine kidney (Asselin and Marcu, 1989; Cowley et al., 1989), and regenerating liver (Thompson et al., 1986; Beer et al., 1987) have provided further correlations between c-myc expression and cell proliferation. Studies of c-myc expression during development, in contrast to those in adult tissues, d o not provide a strong correlation between proliferation rate and mRNA levels. Only a limited subset of dividing embryonic cells expresses high levels of c-myc during human and murine early embryonic development (PfeiferOhlsson et al., 1985; Downs et al., 1989; Hirvonen et al., 1990). However, at later stages of embryogenesis high c-myc expression correlates with cellular proliferation (Schmid et al., 1989). These data indicate that c-myc expression may also contribute to cell migration and/or invasiveness in addition to proliferation during embryogenesis. While c-myc is expressed at high levels in several embryonic and fetal tissues, high-level expression of N-myc and L-myc is more restricted with respect to tissue and the stage of development. The expression of N-myc is very high in the early embryonic period in various tissues and declines dramatically during later development (Jakobovits et al., 1985; Zimmerman et al., 1986). In developing murine embryos, N-myc is expressed in the brain, eye, kidney, lung, heart, and intestine, while L-myc is expressed in the brain, kidney, and lung (Zimmerman et al., 1986; Semsei et al., 1989; Hirning et al., 1991). In the developing human embryo L-myc expression is also detected in fetal skin, spleen, thymus, pancreas, and muscle but the expression ceases after birth (Hirvonen et al., 1990). N-myc expression is higher in pre-B cell lines than in mature B cell lines, while L-myc expression is not found in B cells (Zimmerman et al., 1986). In general, N-myc is expressed in a subset of cells that is at an early stage of differentiation, and the further differentiation of these cells correlates with N-myc downregulation. With the exception of adult lung, L-myc expression is similarly restricted to early stages of a subset of tissues (Zimmerman et al., 1986). Neither N-myc nor L-myc expression correlates well with proliferation in various embryonic cell types, lending further support to the notion that their expression is a characteristic of the undifferentiated state rather than linked to cell growth and division (Mugrauer et al., 1988; Hirvonen et al., 1990). B-myc is expressed in lung, kidney, brain, heart, and spleen with the highest expression in brain (hence B-myc). Both c- and B-myc were found to be expressed in the same tissues but the relative levels differed during development (Ingvarsson et al., 1988). Taken together, these findings indicate that alterations in the expression of

Myc Proteins

141

specific myc family genes may be important in differentiation processes in many different cell types (see DePinho et al., 1991).

C. Effects of c-Myc on Differentiation A rapid downregulation of myc expression is observed in many cell lines following exposure to differentiation inducers (Reitsma et al., 1983; Lachman and Skoultchi, 1984; Griep and DeLuca, 1986; St. Arnaud et al., 1988; see Fig. 5 ) . Constitutive expression of c-Myc prevents cells from leaving the cell cycle (Freytag, 1988), thereby inhibiting differentiation of a number of cell lines including MEL, F9, and 3T3-Ll cells (Coppola and Cole, 1986; Dmitrovsky et al., 1986; Onclercq et al., 1989; Freytag et al., 1990). In addition, antisense inhibition of rnyc expression in proliferating HL-60 and MEL cells leads to growth arrest and induction of terminal differentiation (Griep and Westphal, 1988; Holt et al., 1988; Prochownick et al., 1988). In agreement with these findings, differentiation of U-937 monoblastic cells into macrophages was inhibited by v-Myc expression (Larsson et al., 1988). However, together with one of several differentiation inducing agents, interferon-y can bypass the effects of constitutive v-Myc expression implying that downregulation of myc is not obligatory for U-937 differentiation (Oberg et al., 1991). Adipogenesis in 3T3-Ll cells is regulated by the relative levels of Myc and C/EBPa (Freytag and Geddes, 1992). Constitutive c-Myc expression inhibits differentiation by preventing normal induction of C/EBPa, while its enforced expression overcomes the Myc-induced block of differentiation (Freytag and Geddes, 1992). Furthermore, c-Myc can inhibit myogenic differentiation induced by either MyoD or myogenin independently of Id (Miner and Wold, 1991). Similarly, c-Myc inhibits the differentiation of primary quail myoblast whereas a mutant with a deletion of the C-terminal seven amino acids, including the last leucine residue of the Zip domain, was no longer able to interfere with the differentiation program (La Rocca et al., 1994). Since this Myc mutant is still capable to transform these findings indicate that the transforming function of c-Myc can be distinguished from the Myc-induced block of differentiation. There are also examples where c-myc expression is detectable in some terminally differentiated cells, such as keratinocytes (Dotto et al., 1986) and lens cells (Nath et al., 1987). However, rnyc expression is generally low o r undetectable in many differentiated adult tissues, consistent with the notion that its expression correlates with cell proliferation. This hypothesis is also suggested by Ep-myc transgenic mice where the pre-B cell population is expanded with a simultaneous reduction in mature B cells (Langdon et al., 1986). Proliferation and differentiation represent alternative and mutually exclu-

I42

Marie Henriksson and Bernhard Luscher

sive pathways for cells and there is compelling evidence that Myc may function a t a pivotal control point in the decision-making process.

D. mrgeted Disruption of the c- or N-myc Genes A c-myc null mutation causes lethality in homozygous mice between Days 9.5 and 10.5 of gestation (Davis et al., 1993). Thus, although c-myc expression is associated with dividing cells, it does not appear to be required for proliferation in embryonic stem (ES) cells or in the early embryo. One explanation could be that other family members can replace c-Myc function early in development but not later in embryogenesis, resulting in lethality. Because myc is rapidly induced in response to several growth factors one model could be that c-Myc accelerates the rate at which events involved in cell division occur. According to this model c-myc-deficient cells would be able to proliferate sufficiently to populate the embryo but would be unresponsive to growth factor signaling and concomitantly lacking a proliferative burst necessary for normal development. The c-myc-l- homozygous embryos were generally smaller and retarded in development compared with their littermates (Davis et al., 1993). Abnormalities included heart, pericardium, neural tube, and delay or failure in turning of the embryo. Heterozygous females exhibited reduced fertility. Taken together, this demonstrates that c-myc is necessary for embryonic survival beyond Day 10.5 of gestation; however, it appears to be dispensable for cell division both in ES cell lines and in the embryo before that time. By targeting the N-myc gene in ES cells, a leaky mutation resulting in reduced levels of normal N-myc transcripts was obtained (Bernelot Moens et al., 1992). Mice homozygous for this mutation died immediately after birth owing to an inability to oxygenate their blood. Homozygous mutant embryos were slightly smaller than normal and had a reduction in the size of the spleen, whereas other tissues appeared normal (Bernelot Moens et al., 1992). The lack of other phenotypic defects might be explained by the fact that the leaky mutation produces sufficient levels of N-myc transcripts in other lineages. Three other studies showed that N-myc-l- mice die around Day 11.5 of gestation (Sawai et al., 1991; Charron et al., 1992; Stanton et al., 1992). The homozygous mice appeared to develop normally until the onset of organogenesis. The analysis of c-myc in these mice suggested that it may compensate for the lack of N-myc during early development. With the onset of organogenesis the tissue-specific expression of the different myc family members may become so tightly regulated that compensatory mechanisms are no longer possible. The absence of expression of all myc family members appears most likely to be incompatible with normal tissue development.

143

Myc Proteins

E. Effects of

Max o n Differentiation

The initial analysis of max indicated that its expression, in contrast to c-myc, is rather constant. Equivalent levels of max have been found in quiescent, mitogen-stimulated, and cycling cells (Berberich et al., 1992; Blackwood et al., 1992). In addition, little regulation of rnax expression was observed in differentiating myeloid cell lines such as U-937, HL-60, and ML-1 (Larsson et al., 1994; see Fig. 5). However, recent findings indicate that rnax mRNA and protein are downregulated during erythroid differentiation (Dunn et a)., 1994; Delgado et al., 1995). Similarly, a 10-fold decrease in Max was observed during differentiation of F9 embryonal carcinoma cells (B. Liischer, unpublished observation). Furthermore, max expression has been shown to be growth regulated in epithelial cells (Martel et al., 1995). These findings clearly indicate that max is regulated at the transcriptional level, but because the protein has a very long half-life this regulation is unlikely to have short-term consequences. To determine if overexpression of max could influence differentiation, wild-type Max and a basic region mutant of Max were introduced into MEL cells. Significant overexpression of wild-type Max resulted in reduced growth, and HMBA-mediated differentiation was delayed (Cogliati et al., 1993). In contrast, the mutant Max exhibited growth retardation in GO/G1 and spontaneous differentiation (Cogliati et al., 1993). Furthermore, this mutant was also able to overcome a c-Myc block of differentiation, most likely by absorbing c-Myc into complexes incompetent for DNA binding (S. Segal, personal communication). In order to understand the different effects of Max on both cell growth and differentiation, it should be remembered that overexpression of Max does not only influence Myc function but will most likely also interfere with the role of Mad family proteins. Similar to the previously described findings, a basic region mutant of Max, dMax, has been shown to act as a transdominant negative repressor of Myc DNA binding and was able to revert N-Myc-induced changes in neuroblastoma gene expression (Billaud et al., 1993). Again it was suggested that the biological consequences are a result of sequestering of N-Myc into nonDNA-binding N-Myc/dMax complexes. As far as has been analyzed, Max is essential for c-Myc function. Support for the important role of Max came from recent observations that the homozygous deletion of rnax is embryonically lethal. m a x - / - embryos start to die at some point between Days 3.5 and 6.5 of gestation (H.-W. Lee and R. DePinho, personal communication). max-deficient embryos therefore arrest before the c-myc null embryos (Davis et al., 1993). A likely explanation for this is that while the different Myc proteins might compensate for each other, Max has no obvious homologue and its loss may result in the absence of any Myc function. The broad time range of embryonal death could be a

I44

Marie Henriksson and Bernhard L u s c h e r

reflection of the long half-life of Max in that maternal proteins may support initial growth. These findings further emphasize that Myc function is essential for growth and development.

F. Expression Pattern of the mad Family Genes During organogenesis in mouse embryos mad mRNA was predominantly expressed in the liver and in the mantle layer of the developing brain (Vastrik et al., 1995a). At later stages expression was detected in neuroretina, epidermis, and whisker follicles. In adult mice mad was expressed at variable levels in most organs analyzed. The analysis of epidermal keratinocytes and the intestine showed that mad expression is associated with the more differentiated cells (Hurlin et al., 1995b; Vastrik et al., 1995a). Furthermore, downregulation of myc- genes occurs concomitant with upregulation of mad family genes in the developing central nervous system and the epidermis (Hurlin et al., 1995a). mad levels are very low or not detectable in proliferating cells but are rapidly induced upon the induction of differentiation in hematopoietic cell lines as well as in primary human keratinocytes (Ayer et al., 1993; Larsson et al., 1994; Hurlin et al., 1995b). The transcription of the gene is paralleled by the appearance of Mad and the formation of Mad/Max heterodimers (Ayer and Eisenman, 1993; Hurlin et al., 1995b; see Fig. 5). Keratinocytes transformed with HPVl8 vary in their ability to differentiate and Mad expression was only detected in those cultures retaining the potential to differentiate (Hurlin et al., 1995b). Taken together, these data demonstrate that mad is expressed in nonproliferating, terminally differentiated cells of certain tissues (Ayer et al., 1993; Larsson et al., 1994; Hurlin et al., 1995a,b; Vastrik et al., 1995a). In addition, a constant or increasing level of mxil was observed with progressive development and growth arrest in many organs of the mouse (Schreiber-Agus et al., 1995). m x i l shows widespread tissue expression, with a preference for heart, brain, and lung: tissues in which the cells are terminally differentiated (Zervos et al., 1993). mxil is unique among the mad family genes in that it is expressed both in proliferating as well as in differentiating cells of the developing central nervous system and the epidermis (Hurlin et al., 1995a). In analogy with mad, expression of mxil has been shown to be induced upon induction of differentiation of hematopoietic cells (Zervos et al., 1993; Larsson et al., 1994). These findings have led to the suggestion that c-Myc/Max complexes are replaced by Mad/Max or M x i l / M a x complexes during the differentiation process and/or inhibition of cell growth (Fig. 5). Recent detailed analysis of the different protein complexes in differentiating U-937 cells revealed that Myc/Max, Max/Max, and Mad/Max dimers coexist, with a preference for Myc/Max in undifferenti-

Myc Proteins

I45

ated cells and Mad/Max in differentiated cells. Furthermore, all three complexes are DNA-binding competent (L.-G. Larsson and B. Luscher, unpublished observation). At present, it is not known at which point during differentiation Mad and/or Mxil are functionally important. One possibility is that these proteins are relevant for the arrest of cell growth in response to differentiationinducing agents. The rapid induction of Mad would be in favor of such an interpretation. In addition, Mad and/or M x i l may function by inducing and maintaining a differentiated phenotype which is compatible with their expression throughout the differentiation process. Recent data implicate Mad and M x i l in the former without excluding the latter. Mad- and M x i l expressing cells appear to grow slower than control cells (Cerni et al., 1995; Chen et al., 1995; Schreiber-Agus et al., 1995) and microinjection of Madexpressing plasmids into quiescent fibroblasts inhibits serum-induced S phase entry (A. Menkel and B. Luscher, unpublished observation). Furthermore, ectopic expression of Mad also interferes with CSF-1-induced proliferation by inhibiting the progression from G 1 to S phase (M. Roussel, D. Ayer, and R. Eisenman, personal communication). These findings indicate potent roles of Mad and probably M x i l in interfering with cell growth which may be a prerequisite for differentiation.

VI. MYC-MEDIATEDAPOPTOSIS Apoptosis o r programed cell death is an important mechanism to balance cell proliferation and to remove unwanted cells during the development and homeostasis of multicellular organisms (Vaux et al., 1994; Steller, 1995; Thompson, 1995). Whereas it has been evident for many years that cell growth requires the action of mitogenic factors, it has only recently been realized that cells also need signals to survive (Raff et al., 1993). If survival factors are in limited supply cells will compete for them. Cells which d o not receive sufficient amounts of survival signals will induce a cell autonomous program leading to cell death by apoptosis. This process may also be triggered specifically, e.g., by stimulating the death program through cell surface structures, including receptors of the Fas or T N F type. Apoptosis is characterized by shrinkage of the cell, membrane blebbing, chromatin condensation, frequently by DNA fragmentation, and finally the appearance of apoptotic bodies which are then phagocytosed by surrounding cells (see Wyllie, 1985). In contrast to cell death through necrosis, no leakage of cellular contents occurs, preventing an inflammatory response. In recent years it has been recognized that the regulation of apoptosis is also critical for tumor development. In addition to positive signals stimulat-

146

Marie Henriksson a n d Bernhard Luscher

ing growth, cells progressing from a normal to a malignant phenotype need to inhibit apoptosis, which may occur as a consequence of inappropriate growth signals o r genomic instability. Activated forms of protooncoproteins can provide an inappropriate, constitutive growth signal which may lead to a conflict of interests when a cell needs to slow or arrest growth, such as after DNA damage. In normal circumstances, such a conflict would be resolved by the activation of an apoptotic program leading to the elimination of the respective cell. The acquirement of genetic changes leading to both constitutive growth stimulation and inhibition of apoptosis therefore provides a selective advantage for tumor growth (see Fischer, 1994; Lowe et al., 1994).

A. Overexpression of c-Myc Triggers Apoptosis A number of observations have suggested that the levels of c-Myc correlate with susceptibility to apoptosis. This is somewhat paradoxical because c-Myc has been strongly associated with proliferation. One of the first observations associating c-Myc with cell death was obtained through efforts to overexpress this protein in CHO cells. Stable transfectants with a heat-shock promoter-c-myc construct underwent cell death after induction of c-Myc synthesis by heat-shock treatment (Wurm et al., 1986). Furthermore, increased rates of apoptosis have been observed in myc-transformed fibroblasts (Wyllie et al., 1987). In addition, it was shown that constitutive expression of c-Myc results in accelerated apoptosis in the myeloid cell line 32D upon IL-3 withdrawal (Askew et al., 1991). More direct evidence for an important role of c-Myc in the induction of apoptosis was demonstrated by the use of conditional Myc constructs. Serum-starved Ratl cells expressing c-MycER were shown to undergo apoptosis upon addition of p-estradiol o r hydroxytamoxifen (Evan et al., 1992; Harrington et al., 1994a). Similar observations were made in some other cell lines including NIH3T3 and mouse embryo fibroblasts (Hermeking and Eick, 1994; Wagner et al., 1994). In support of the concept that Myc is involved in regulating apoptosis, parallel studies demonstrated that c-Myc is required for T cell receptor (TCR)-stimulated apoptosis in T cell hybridomas (Shi et al., 1992). Together these studies provided evidence for an important role of c-Myc in the induction of cell death. However, not every cell line is sensitive to Myc overexpression. Upon infection of mouse L929 cells with a v-myc-carrying retrovirus and exposure to low serum, no apoptosis was observed (Facchini et al., 1994). Fusion of L929 with R a t l cells restored the sensitivity to Myc, suggesting that loss of function of one or several components required for Myc-induced apoptosis had occurred in L929 cells. At present the nature of these components is

Myc Proteins

I47

unclear, but p53 is a potential candidate because it has been shown to be required for Myc-induced apoptosis (see below). The relevant regions in Myc for apoptosis induction comprise the TAD and the bHLHZip domain, the same structures important for all other biological functions of c-Myc, including S-phase progression and transformation (Evan et al., 1992; Goruppi et al., 1994). Not only is the bHLHZip region relevant but the ability to interact with Max is essential for apoptosis, both in Myc-overexpressing fibroblasts after serum removal and in T cell hybridomas after challenging with anti-TCR antibodies (Amati et af., 1993b; Bissonnette et al., 1994a). These data would argue that c-Mycdriven apoptosis is mediated by c-MycIMax-specific transactivation. It has been suggested that cyclin A and ODC participate in Myc-induced apoptosis (Hoang et al., 1994; Packham and Cleveland, 1994). The ODC gene is of interest because it has been suggested to be a direct c-Myc target (see Section IV,C). ODC catalyzes the rate-limiting step in polyamine synthesis and is therefore required for S phase entry (Bowlin et al., 1986; Heby and Persson, 1990). In addition, this enzyme can cooperate with Ras in the transformation of NIH cells (Hibshoosh et al., 1991). The forced expression of ODC in 32D myeloid cells made these cells sensitive to IL-3 withdrawal, resulting in enhanced apoptosis similar to the findings in 32DIc-Myc cells (Packham and Cleveland, 1994). If ODC is a relevant downstream target for c-Myc-induced cell death, one may expect that interfering with ODC function would inhibit Myc-driven apoptosis. Indeed, it was observed that the blocking of ODC resulted in inhibition of apoptosis in Myc-overexpressing cells (Packham and Cleveland, 1994). Forced expression of cyclin A, a gene induced by Myc overexpression, was itself sufficient to induce apoptosis in RatlA cells under low serum conditions (Hoang et al., 1994). Both ODC and cyclin A may therefore be downstream of c-Myc in the apoptosis pathway (see also discussion below).

B. REGULATION OF MYC-INDUCEDAPOPTOSIS I . BcI-2 AND p53 Among the proteins relevant for apoptosis, Bcl-2 is of particular interest because it inhibits many, yet not all, types of cell death (Reed, 1994). Bcl-2 resides in the nuclear envelope, parts of the endoplasmic reticulum, and in the outer mitochondria1 membrane (Reed, 1994 and references therein). The function of Bcl-2 is not well defined but appears to be essential. Bcl-2deficient mice complete embryonic development but display growth retardation and early postnatal mortality (Veis et al., 1993). Hematopoiesis, includ-

I48

Marie Henriksson and Bernhard Liischer

ing B and T cell differentiation, was initially normal; hence, Bcl-2 activity is not absolutely required for the development of those lineages. However, over time bcl-2-/- mice developed massive apoptosis within the thymus and the spleen concomitant with an almost complete loss of lymphocytes (Veis et al., 1993). Consequently, Bcl-2 may have its most significant role in maintaining homeostasis in adult tissues (Korsmeyer, 1995). Because Bcl-2 has also been implicated in tumorigenesis (see Section VII,C) it was tested for inhibition of c-Myc-induced apoptosis. In both Rat1 and RatlA fibroblasts Bcl-2 protected cells from c-Myc-induced apoptosis (Fanidi et al., 1992; Wagner et al., 1993b). Similar conclusions were drawn for C H O cells overexpressing c-Myc (Bissonnette et al., 1992). In this system, Mcl-1, a recently identified member of the Bcl-2 family, showed a similar effect to Bcl-2 (Kozopas et al., 1993; Reynolds et al., 1994). These findings suggest that apoptosis induced by deregulated expression of c-Myc follows common pathways. The tumor suppressor protein p53 is another important molecule implicated in regulating apoptosis. p53 has been suggested to be a molecular switch activated after DNA damage leading either to growth arrest or to apoptosis (see Selivanova and Wiman, 1995). p 5 3 - l - mice show an increased tumor rate, probably as a consequence of an inability to respond appropriately to DNA damage resulting in the accelerated accumulation of mutations (Donehower et al., 1992; Jacks et al., 1994). Because a number of tumor cell lines with deregulated c-myc contain a mutated or deleted p.53 gene (see below) it has been speculated that this protein could play a role in Myc-induced apoptosis (Wagner et al., 1994). In addition, p53 was found to be induced upon activation of a c-MycER construct in serum-starved fibroblasts (Hermeking and Eick, 1994). p53 accumulation was also detected in myclras-transformed cells (Lu et al., 1992). Despite increased abundance of p53, c-Myc is capable of driving cells into S phase (Hermeking et al., 1995). To test if functional p53 is required for c-Myc-induced apoptosis, c-MycER constructs were introduced into p53-1- mouse embryo fibroblasts (MEF). Upon activation of c-MycER by p-estradiol, serum-starved cells no longer underwent apoptosis but still induced DNA synthesis (Hermeking and Eick, 1994; Wagner, et d., 1994). Furthermore, a p 5 3 - / - MEF line stably expressing a temperature-sensitive p53 mutant and c-MycER was sensitive to p-estradiol-induced apoptosis only at the permissive temperature with p53 being in the wild-type configuration (Wagner et al., 1994). In agreement with these findings SV40 T antigen, which can bind to p53 and thereby inactivate it, was found to inhibit Myc-induced apoptosis (Hermeking et al., 1994). These experiments demonstrated that p53 is obligatory for Myc-induced apoptosis. If c-Myc is such an efficient inducer of apoptosis the question remains as to how tumors and tumor cell lines with elevated Myc expression escape

Myc Proteins

I49

apoptosis. One explanation is provided by the observation that a large proportion of tumors with deregulated Myc expression either have deleted o r mutated p53 o r overexpress Bcl-2 (Farell et al., 1991; Gaidano et al., 1991; Wiman et al., 1991; Reed, 1994). This suggests that apoptosis is of considerable importance as a selective factor in the evolution of Myc-overexpressing cells. A survey of a large panel of B and T cell tumors and tumor cell lines showed that mutations in p53 are frequent in BLs and in B-ALL (L3type), tumors with consistent overexpression and/or deregulated c-myc expression, but not in other tumors (Farell et al., 1991; Gaidano et al., 1991; Wiman et al., 1991). Apoptosis in BLs appears to require c-Myc and can be suppressed by Bcl-2 and Epstein-Barr virus (EBV) latent proteins (Gregory et al., 1991; Henderson et al., 1991; Milner et al., 1992, 1993). EBV is associated with endemic and with approximately 3 0 % of sporadic BLs. It is possible that EBV infection provides a selective advantage by inhibiting apoptosis. EBV has at least two mechanisms to modulate apoptosis. The latent membrane protein LMPl has been shown to induce Bcl-2 expression, whereas BHRFl encodes a Bcl-2-related protein (Henderson et al. 1991, 1993; Finke et al., 1992). The evidence summarized previously and under Section VII,C shows a strong correlation between Myc’s ability to transform cells and to induce apoptosis. It therefore seems that c-Myc-dependent transformation can be enhanced by interfering with apoptosis. This is at least one of the functions in which Bcl-2 and p53 (its loss) are implicated. An immediate question arising is whether other Myc-cooperating proteins exert a similar action. Interestingly, it has been suggested that Ras, Abl, and Pim-1 may also affect apoptosis negatively (Wyllie et al., 1987; Evans et al., 1993; Moroy et al., 1993; Bissonnette et al., 1994b; McGahon et al., 1994). Thus, a pattern emerges where proteins cooperating with Myc in transformation inhibit apoptosis.

2. SURVIVAL FACTORS As indicated previously, apoptosis is antagonized by survival factors. The findings that serum withdrawal was required to induce apoptosis in Mycoverexpressing fibroblasts and that withdrawal of IL-3 triggered cell death in myeloid cells with constitutive c-Myc expression suggested that survival factors could also play an important role under these experimental conditions. Insulin-like growth factors, and platelet-derived growth factor, but not a number of others, including epidermal growth factor (EGF), were identified as survival factors for c-Myc-overexpressing cells (Harrington et al., 1994a). Furthermore, these factors appear to work in any phase of the cell cycle, which is of interest because fibroblasts d o not require serum to complete the mitotic cycle once they have passed the restriction point in late G1.

150

Marie Henriksson a n d Bernhard L u s c h e r

In addition, this separates the mitotic from the antiapoptotic activities of these molecules. At present, it is unclear why other mitogenic factors such as EGF or bombesin do not exhibit protective activity. It will be of interest to establish the relevant downstream signaling pathways leading to inhibition of apoptosis.

3. C-Myc MODULATES APOPTOSIS INDUCED BY TUMOR NECROSIS FACTOR 01 Tumor necrosis factor a (TNF) has multiple biological activities, including induction of apoptosis in some cell lines, whereas others are not sensitive to the toxic action of TNF (for review see Larrick and Wright, 1990; Fiers, 1991). Interestingly, sublethal doses of TNF have been found to protect cells from the subsequent action of TNF, indicating that it can activate genes required for both activation and inhibition of apoptosis. TNF-induced genes included manganous superoxide dismutase (MnSOD), plasminogen activator inhibitor type 2 (PAI-2),A20 zinc-finger protein, and heat-shock protein 70, all of which individually can confer resistance to TNF (Wong et al., 1989; Kumar and Baglioni, 1991; Jaattela etal., 1992; Opipari etal., 1992). Because the adenovirus E1A protein can render cells sensitive to the action of TNF, c-Myc was tested for its involvement in the TNF response. c-Mycoverexpressing fibroblasts were found to be more sensitive to TNF-induced apoptosis than control cells, suggesting that c-Myc could enhance the TNF effect (Klefstrom et al., 1994). This was not accompanied by any alteration in the cell cycle distribution of the treated cells, indicating a specific effect on apoptosis. Both Bcl-2 and MnSOD inhibited TNF/c-Myc-induced apoptosis. The generation of free oxygen radicals may be relevant in Myc-induced apoptosis in low serum because a small protective effect by MnSOD was observed. A second study showed that TNF induced the expression of c-Myc in a TNF-sensitive but not in a -resistant HeLa cell clone, without detectable differences between the two cell lines in the expression of several known TNF-regulated genes (Janicke et al., 1994). Antisense c-myc rendered the sensitive cell line more resistant, supporting the concept that Myc may be a downstream effector of TNF, i.e., implicating c-Myc as an important mediator in TNF-induced apoptosis.

4. C-MyC-INDEPENDENT APOPTOSIS The studies described previously indicate that Myc can drive apoptosis and that its expression is required in some systems for efficient induction of cell death. However, there are examples in which Myc does not seem to be important. For instance, FDC-P1 cells expressing Bcl-2 cease to grow after 1L-3 withdrawal, downregulate c-myc, and stay alive for extended periods of time (Vaux and Weissman, 1993). After turning off bcf-2 expression these

Myc Proteins

151

cells rapidly undergo apoptosis without apparent c-myc transcription. In addition, c-myc downregulation was proposed to be important during glucocorticoid-induced apoptosis of human leukemia cells (Thulasi et al., 1993). These findings indicate that in certain circumstances c-Myc expression is not obligatory for cell death. Clearly there is still much to learn regarding the requirements of c-Myc-mediated apoptosis in different cell types and in response to various signals.

C. How Does c-Myc Modulate Apoptosis? The data summarized in this section demonstrate quite clearly that Myc’s function in apoptosis cannot be separated from its role in promoting cell growth and inducing transformation. Because the findings implicating c-Myc in apoptosis were somewhat unanticipated it seems worthwhile to ask whether this is unique to Myc, or whether other growth-promoting gene products can exert similar effects. It has become evident that in addition to c-Myc, a number of proteins, including c-Myb, B-Myb, cyclin A, E2F, and ODC, as well as the viral oncoproteins E1A and E7, can accelerate apoptosis (Rao et al., 1992; Hoang et al., 1994; Howes et al., 1994; Packham and Cleveland, 1994; Pan and Griep, 1994; Selvakumaran et al., 1994; Shan and Lee, 1994; Smarda and Lipsick, 1994; Bies and Wolff, 1995). In common with c-Myc, all these proteins can positively affect cell growth by driving cells into S phase and/or transform cells. Thus, it appears that dominant effects on cell growth may be frequently combined with the ability to induce apoptosis. What is the basis for these opposing effects? One possible model is that the constitutive (over)expression of these proteins results in a conflict of interest within the cell. On the one hand, the G1-S restriction point is overcome and subsequently S phase is induced and, on the other hand, appropriate signals normally required to enter S, such as survival factors, may be missing. This conflict could be resolved by the initiation of an endogenous suicide program. Little is known about the regulation of the previously described findings. However, for c-Myc p53 plays a critical role. The activation of c-MycER leads to the induction of both p53 transcription and protein stability, which in turn results in p21 accumulation (Hermeking and Eick, 1994; Wagner et al., 1994). Despite the presence of p53 and p21, which are potent inhibitors of cell cycle progression, arresting cells in late G1, constitutively expressed c-Myc is able to drive cells into S phase (Hermeking and Eick, 1994; Wagner et al., 1994). Such cells will undergo apoptosis even in the presence of serum if wild-type p53 is synthesized constitutively (Wagner et al., 1994). These data suggest that serum or survival factors may function in preventing the activation of endogenous p53 by c-Myc and consequently apoptosis will be

152

Marie Henriksson and Bernhard Luscher

inhibited. Therefore, a possible scenario in the absence of survival factors may involve two successive modes of action for p53 in response to overexpressed c-Myc. First, induction of p21 to prevent progression into S phase, and second, stimulation of an apoptotic program if the first function has become obsolete. The concept that S phase may be important for Mycinduced apoptosis is also supported by the above described finding that O D C function is required for Myc-induced apoptosis, since blocking O D C prevents S phase entry. As an alternative to the previously described conflict model, it has been suggested that c-Myc may constitutively activate the apoptotic pathway as a normal physiological function (Harrington et al., 1994b). This would be counteracted by survival factors, and their availability would be an important determining factor for cell fate. Both options described are possible at the molecular level. The identification of Myc target genes directly involved in the apoptotic process will be of utter importance in resolving this issue. The p53 gene may be such a target because it has been suggested to be activated by c-Myc (Reisman et al., 1993; Hermeking and Eick, 1994; Roy et al., 1994). In addition, the protein appears to be stabilized in response to c-Myc overexpression (Hermeking and Eick, 1994), indicating that Myc may regulate p53 by two different pathways. It remains to be determined if p53 is regulated by c-Myc under normal physiological conditions or if overexpression of c-Myc is a prerequisite. Furthermore, it will be important to test if the other proteins mentioned, which also can induce apoptosis, are working through p53. This information will be relevant in understanding the interrelationship between growth and apoptosis.

VII. EFFECTS OF Myc NETWORK PROTEINS

ON TRANSFORMATION

A. Alterations of

c-myc in Tumors

The analysis of a number of chicken retroviruses carrying myc sequences demonstrated the potency of the v-myc gene in inducing a variety of different tumor types. Subsequent work showed that the c-myc locus is altered in a large number of animal as well as human tumors (for reviews see Field and Spandidos, 1990; DePinho et al., 1991; Spencer and Groudine, 1991; Marcu et al., 1992). Chromosomal translocations involving c-myc and one of the immunoglobulin loci are characteristic of mouse plasmacytomas, rat immunocytomas, and Burkitt lymphomas. c-myc translocations also occur in human B and T cell acute lymphocytic leukemias (ALL). Increased c-myc expression due to gene amplification has been found in neoplasms derived

Myc Proteins

153

from a wide variety of tissues; small cell lung carcinoma, breast carcinoma, osteosarcoma, colon carcinoma, glioblastoma, cervix carcinoma, myeloid leukemia, and plasma cell leukemia. In addition to these genomic alterations, a different mechanism altering c-Myc expression involving protein stabilization has been described in human glioma cell lines (Shindo et al., 1993). These studies suggested that c-Myc frequently participates in tumor development. What is common to all these tumors is the inability to efficiently downregulate c-myc expression in response to differentiating agents. This results in a reduced capacity of these cells to differentiate and therefore in an increased potential to cycle. Such cells may be the targets for additional somatic mutations potentially leading to tumor formation. These predictions have since been strongly supported by a large number of experimental data obtained in animal models as well as from tissue culture cells.

B. Tumorigenesis in c-myc Transgenic Mice Transgenic mouse models allow the assessment of the function of certain genes of interest in normal development and tumorigenesis in a living organism. Depending on the chosen promoter-enhancer linked to the gene of interest, its expression, and hence, biological effects, can be confined to particular cell lineages. Transgenic mice have been an invaluable tool in demonstrating tissue-specific transforming activity of oncogenes, have provided insight into the degree of their transforming potential, and have allowed the assessment of oncogenic cooperativity. In early studies transgenic mice were generated that expressed the myc gene driven by the mouse mammary tumor virus (MMTV) long terminal repeat (LTR), a promoter inducible by glucocorticoid hormones. The mice developed mammary adenocarcinomas during one of their early pregnancies (Stewart et al., 1984). Importantly, however, the substantial latency and the clonality of the tumors indicated that the MMTV-myc transgene represented only one of several steps in establishing the disease. This was substantiated by the observed synergistic action between c-myc and activated Ha-ras in transgenic mice that possessed both genes under the control of the MMTV LTR (Sinn et al., 1987). These double transgenics showed a dramatic acceleration of the development of mammary carcinomas compared to the single transgenic animals. However, these tumors arose in a stochastic fashion and appeared to be monoclonal, indicating that additional somatic events were still necessary for full malignant progression (Sinn et al., 1987). Transgenic mice overexpressing the c-myc gene within the B lymphocyte compartment by virtue of the presence of the immunoglobulin heavy chain enhancer (Ek) in the promoter region have been instrumental in studying the role of c-Myc in B cell lymphomagenesis (Adams et a/., 1985; Harris et al.,

I54

Marie Henriksson and Bernhard Liischer

1988). The constitutive c-myc expression promotes a benign polyclonal overproduction of cycling pre-B cells and a reciprocal reduction in the number of mature B cells (Langdon et al., 1986), possibly due to a partial block of differentiation. Although these mice develop pre-B or B cell lymphomas spontaneously, the tumors are clonal and appear only after a variable latency period, indicating that constitutive c-myc expression predisposes for, but is not sufficient to provoke, B lymphoid tumors (Adams et al., 1985; Harris et al., 1988). Pre-B cells of Ep-c-myc mice did not show altered growth requirements in vitro but acquired a malignant phenotype upon the introduction of v-Ha-ras or v-raf (Alexander et al., 1989a). Furthermore, analysis of spontaneous secondary events revealed that 2 out of 14 Ep-myc-induced lymphomas carried a mutated ras gene (Alexander et al., 1989b). In addition, infection of the Ep-myc mice with retroviruses containing either v-ras o r v-raf accelerated the onset of lymphomagenesis (Langdon et al., 1989). Taken together, these results indicate that the likelihood of accumulating somatic mutations in genes that cooperate with myc in oncogenesis increased as a consequence of the expanded pre-B cell population (Langdon et al., 1986; Harris et al., 1988). Experimental support for this hypothesis came from elegant studies in which the gene encoding the membrane-bound immunoglobulin heavy (IgH) chain was used (Nussenzweig et al., 1988). The presence of an already assembled IgH chain is believed to accelerate B cell maturation, thereby reducing the pre-B cell compartment. Indeed, tumorigenesis was suppressed in Ep-c-myc mice by coexpression of a functionally rearranged immunoglobulin transgene, indicating that the increased population of vulnerable pre-B cells is an important determinant for tumor formation in Ep-c-myc mice (Nussenzweig et al., 1988). Ep-N- and Ep-L-myc transgenic mice also developed lymphoid malignancies although with a lower incidence and a longer latency period compared to Ep-c-myc animals (Dildrop et al., 1989; Rosenbaum et al., 1989; Moroy et al., 1990). The directed expression of the max gene to lymphoid cells by means of an immunoglobulin heavy chain enhancer did not alter the composition of lymphoid cell populations in adult animals although the max levels were substantially higher than normal (Lindeman et al., 1995). In addition, these mice were not predisposed to lymphomas. Analysis of Ep-c-myclEp-max double transgenic animals revealed that max overexpression attenuated the premalignant B lymphoproliferative state induced by the Ep-c-myc transgene and reduced the rate of lymphoma onset. This was attributed to the observed decrease in the number of cycling pre-B cells hence reducing the number of targets for additional mutations cooperating with myc in tumorigenesis (Lindeman et al., 1995). These results indicate that the relative levels of c-Myc/Max versus Max/Max may be relevant for B cell growth and development.

Myc Proteins

I55

Work with transgenic animals has therefore complemented the analysis of tumors in the establishment of c-myc as a critical oncogene in tumorigenesis.

C. Cooperating Oncogenes in c-myc Transgenic Mice Oncogenic synergism between known genes in animals can be studied by retroviral transduction of additional oncogenes into transgenic mice or by crossing mice bearing two different transgenes. An alternative approach that has been used for the identification of new cooperating oncogenes is provided by retroviral insertion (van Lohuizen and Berns, 1990). The rationale is that retroviruses that lack an oncogene, such as Moloney murine leukemia virus (MoMuLV), promote tumorigenesis primarily by insertion near or within cellular oncogenes, thereby enforcing their expression and/or altering their structure. The presence of the viral genome provides a “tag” for these genes, thereby facilitating their cloning. MoMuLV infection of E p r n y c transgenic mice results in a dramatic acceleration of lymphomagenesis by retroviral insertion (Haupt et al., 1991; van Lohuizen et al., 1991). Three loci were found to be involved in 75% of these tumors; pim-1, bmi-llbla-1, and pal-1. A number of the lymphomas contained proviruses in more than one locus, suggesting that the activation of more than one additional gene confers a selective advantage (Haupt et a/., 1991; van Lohuizen et al., 1991; van der Lugt, 1995). Examples of oncogenes which synergize with c-myc in transformation will be discussed below. I . a61

The c-abl gene was originally defined as the cellular homologue of the v-abl oncogene of the transforming Abelson murine leukemia virus (AMuLV) (Goff et al., 1980). The gene is ubiquitously expressed and encodes a tyrosine kinase important for cell growth (Rosenberg and Witte, 1988; Wang, 1993). c-Abl resides both in the cytoplasm and in the nucleus and contains a sequence-specific DNA-binding domain (Dikstein et al., 1992). v-Abl and Bcr-Abl are both naturally occurring leukemogenic oncoproteins resembling c-Abl but with activated tyrosine kinase domains (Kurzrock et al., 1988). A-MuLV characteristically induces pre-B cell lymphomas following in vitYO infection of mice (Risser et al., 1982). E p v - a b l transgenic mice, in contrast, exhibited a high predisposition for tumors of the plasma cell, the end stage of B cell differentiation. Interestingly, these plasmacytomas frequently carried a rearranged c-myc gene (Rosenbaum et al., 1990). Furthermore, the crossing between E p v - a b l and E p c - m y c transgenic mice yielded progeny

I56

Marie Henriksson and Bernhard Luscher

with a greatly accelerated onset of tumor formation, confirming the synergistic effect of the two genes in plasmacytomagenesis (Rosenbaum et ul., 1990). The fact that plasmacytomas, but no tumors earlier in the B lineage, arose in these animals indicated a stage-specific oncogenic cooperativity of v-abl and myc. This is supported by the finding that v-rafand v-Ha-ras, but not v-abl, increased the transformation efficiency of E p c - m y c pre-B cell clones (Alexander et al., 1989a). In addition, a retrovirus expressing both v-abl and c-myc solely induced plasmacytomas rather than pre-B cell lymphomas (Weissinger et al., 1993). In this respect it is intriguing that the most striking phenotype of mice that carry a homozygous disruption of c-a61 is an effect on lymphoid development (Schwartzberg et al., 1991; Tybulewicz et al., 1991). These mice had a drastic reduction in B cell progenitors and a less dramatic decrease in developing T cells, implying that Abl plays an important role in the differentiation of lymphoid precursors or, alternatively, in retaining these cells at early differentiation stages (Schwartzberg et al., 1991).

2. 6cl-2 While most known oncoproteins seem to stimulate cell proliferation, Bcl-2 promotes cell survival by blocking apoptosis (Vaux etal., 1988; Reed, 1994; see Section VI). The bcl-2 oncogene was isolated from the breakpoint of a (14;18) chromosomal translocation that is a hallmark of most human follicular B lymphoid tumors and a proportion of diffuse large-cell lymphomas (Bakhshi et al., 1985; Tsujimoto et al., 1985,1987; Yunis et al., 1987). This translocation brings the bcl-2 gene into the IgH chain locus creating a bcl2-lg fusion gene (Cleary et al., 1986; Seto et al., 1988). Transgenic mice carrying either a bcl-2-lg minigene or a E p b c l - 2 gene exhibited a follicular expansion of small resting B cells which accumulated as a result of extended survival rather than increased proliferation (McDonnell et al., 1989, 1990; Strasser et al., 1990a). These cells survived abnormally well when cultured in the absence of cytokines or mitogens due to the prevention of apoptotic death (Strasser et al., 1991). After a long latency period these animals developed lymphomas or plasmacytomas, indicating that the prolonged B cell life span increases tumor incidence (Strasser et al., 1990a; McDonnell and Korsmeyer, 1991). The plasmacytomas derived from both E p b c l - 2 and the bcl-2-Ig minigene mice displayed a high frequency of myc rearrangements presumably contributing to their etiology (McDonnell and Korsmeyer, 1991; Strasser etal., 1993). Infection of pre-B cells from Ep-myc transgenic mice with a bcl-2 retrovirus permitted the eventual outgrowth of immortalized pre-B lines (Vaux et al., 1988). More strikingly, mice expressing both myc and bcl-2 transgenes exhibited hyperproliferation of pre-B and B cells and developed tumors much faster than the myc transgenic animals (Strasser

Myc Proteins

157

et al., 1990b). Therefore, by extending cell survival, Bcl-2 may increase the chance of secondary genetic changes responsible for tumorigenicity. Surprisingly, the tumors in the double transgenics were not derived from the abundant pre-B and B cells as in the EF-myc transgenics (Harris et al., 1988), but from a cell with the hallmarks of primitive hematopoietic origin (Strasser et al., 1990b). Thus, oncogenic cooperation not only accelerates the onset of neoplasia but can also change the phenotype of the malignancy. These data suggest an attractive model for the nature of the cooperativity between bcl-2 and c-myc. In addition to stimulating proliferation c-Myc can, under certain conditions, also induce apoptosis (see Section VI). Consequently, it is conceivable that the concomitant overexpression of Bcl-2 can overcome the apoptotic influence of Myc leading to a further selective advantage. In vitro, Bcl-2 has been shown to inhibit the apoptotic function of Myc without affecting its mitogenic capacity (see Section VI). However, fibroblasts that express both proteins appear nontransformed and do not form foci even though they proliferate without mitogens. This is in contrast to the oncogenic cooperativity between myc and ras, and thus represents a novel type of oncogene synergy. Yet, bcl-2 can complement as in malignant transformation of REFS (Reed et al., 1990). 3. CYCLIN DI

Cyclin D1 is one of the regulatory subunits of cyclin-dependent kinases (CDKs) and is as such a rate-limiting controller of G1 progression in mammalian cells (for review see Hunter and Pines, 1994; Sherr, 1995). The cyclin D1 gene is overexpressed in some human breast, gastric, and esophageal carcinomas due to amplification, and in some human parathyroid adenomas as a result of rearrangements. In addition, cyclin D1 represents that product of the bcl-1 gene which is juxtaposed to the immunoglobulin heavy-chain locus in the t ( 11;14) chromosomal translocation occurring in several human B cell malignancies. EF-cyclin D1 transgenic mice have been generated in order to investigate the effect of deregulated cyclin D1 on lymphocyte development. Despite high transgene expression their lymphocytes were normal in cell cycle activity, size, and mitogen responsiveness, although young transgenic animals contained fewer mature B and T cells. Spontaneous tumors were infrequent but Ep-directed coexpression of cyclin D1 and N-myc or L-myc in double transgenic mice revealed a strong synergistic effect between Myc and cyclin D1 in lymphomagenesis (Bodrug et al., 1994; Lovec et al., 1994a). It has been speculated that this synergy is due to a combined action of the two genes in inhibiting differentiation (Bodrug et al., 1994). At the molecular level, the overexpressed cyclin might contribute to oncogenesis by enhancing the ac-

158

Marie Henriksson a n d Bernhard Liischer

tivity of its CDK partner(s) or by compromising the activity of one o r several of the newly discovered inhibitors of CDKs. In vitro, cyclin D 1 has been shown to cooperate with H a m s in the transformation of primary REFS (Lovec et al., 1994b) but not baby rat kidney (BRK) cells (Hinds et al., 1994). However, cyclin D 1 contributes to transformation of BRK cells by complementing a defective E l A gene and activated Ha-ras (Hinds et al., 1994). In neither of these studies did cyclin D1 and c-myc lead to a transformed phenotype. 4. pim-/

Pim-1 is a cytoplasmic Ser/Thr protein kinase with unknown physiological substrates (Saris et al., 1991). To verify the original observation showing proviral integration near the pim-I locus (Cuypers et al., 1984; Selten et al., 1985) and to exclude that some other, as yet unidentified, gene in the vicinity was important for transformation, E p p i m - I transgenic animals were generated. The Ep-pim-I transgene was expressed at equal levels in both B and T cells, but the animals were clearly predisposed to the development of T cell neoplasia although the tumor incidence was low (van Lohuizen et al., 1989). MoMuLV infection of newborn Ep-pim- I transgenics strongly accelerated T cell lymphomagenesis and either c- or N-myc was found to be activated by proviral insertion in all of the tumors examined (van Lohuizen et al., 1989). Conversely, when Ep-myc transgenic mice were infected with MoMuLV, pim-I was frequently found to be activated, as mentioned previously (Haupt et al., 1991; van Lohuizen et al., 1991). These findings implicated a cooperation between pim-I and myc in lymphomagenesis. In support of this, bitransgenic mice derived from Ep,-c-myc/Ep-pim-I cross-breeding developed pre-B cell leukemia prenatally and died in utero, demonstrating a very strong synergism berween c-myc and pim-1 (Verbeek et al., 1991). However, all tumors described to date are monoclonal, clearly indicating that additional events are required for the development of a fully malignant phenotype. Mice carrying a homozygous deletion of the pim-1 gene exhibited n o obvious phenotype, implicating the presence of a redundantly acting gene(s) (te Riele et al. 1990; Domen et al., 1993a,b; Laird et al., 1993). Indeed, the introduction of a Ep-c-myc transgene into these pim-I null mice and the subsequent infection of the offspring with MoMuLV revealed that more than 8 0 % of the induced tumors carried a proviral insertion in pim-2 (van der Lugt et al., 1995). Because Pim-1 and Pim-2 are approximately 50% homologous at the amino acid level this observation lends further support to the importance of the pim pathway for myc lymphomagenesis (van der Lugt et al., 1995). One indication for the putative molecular function of Pim-1 has been

Myc Proteins

159

obtained by the introduction of a pim-1 transgene in mice homozygous for the lpr mutation (Moroy et al., 1993). The lpr-I- mice lack Fas receptorinduced apoptosis and develop a well-described lymphoproliferative syndrome mainly characterized by the accumulation of abnormal T cells in lymph nodes (Cohen and Eisenberg, 1992; Watanabe-Fukunaga et al., 1992). The pim-1 transgene was shown to accelerate the lymphoproliferation and to inhibit apoptosis indicating a role for pim-1 in the regulation of apoptosis. These results raise the question of whether this reflects the mechanism leading to myc and pim-1 cooperativity. However, in this respect it should be mentioned that pim-1 and bcl-2 also cooperate in the induction of lymphomagenesis (Acton et al., 1992). Although c-myc and pim-1 cooperate in lymphomagenesis in vivo, no demonstration of cooperativity in transformation of cultured cells between pim-1 and c-myc or ras has been observed (A. Berns, personal communication).

5. 6mi-I The bmi-I gene (B cell lymphoma MoMuLV-integration region 1)encodes a nuclear zinc-finger protein believed to be involved in transcriptional regulation (Haupt et al., 1991; van Lohuizen et al., 1991). Distinct domains of Bmi-1 are highly conserved to regions within the Drosophila protein Posterior Sex Combs, a member of the Polycomb group involved in maintaining stable repression of homeotic genes during development. This suggested that Bmi-1 could be involved in the regulation of expression of murine homeotic genes (van der Lugt et al., 1994). bmi-1 -1- mice have a reduced size and exhibit posterior transformation of the axial skeleton, neurological abnormalities, and severe hematopoietic defects (van der Lugt et al., 1994). Epbmi-1 transgenic mice, in contrast, exhibit at least in part the opposite phenotype, an anterior transformation of the axial skeleton (Alkema et al., 1995). Therefore, it seems likely that Bmi-1 is involved in the regulation of murine homeotic genes (Alkema et al., 1995). The overexpression of bmi-1 in Ep-bmi-1 mice perturbs B cell development leading to an expansion of pre-B cells and a block of differentiation as well as a high incidence of lymphomas (A. Berns, personal communication). The synergism between c-myc and bmi-1 in the development of B cell lymphomagenesis indicates that oncogenic cooperation is not restricted to collaborations between nuclear and cytoplasmic oncoproteins, but can involve the concerted action of two nuclear oncoproteins. This cooperation is likely to involve complementary changes in gene expression rather than physical association. One possible mechanism is that Bmi-1 alters the complement of Hox genes that is expressed in hematopoietic cells. Hox genes are good transformers of hematopoietic cells and cooperate well with c-myc (A. Berns, personal communi-

I 60

Marie Henriksson and Bernhard Luscher

cation). Alternatively, Bmi-1 might act on other genes regulating growth and differentiation of lymphoid cells.

D. Assay Systems for In Mtro Transformation The c-myc gene is able to immortalize primary cells in culture; however, deregulated c-myc expression alone is not sufficient to elicit a malignant phenotype. c-myc can cooperate with an activated YUS oncogene in the transformation of rat fibroblasts, rat embryo cells, and murine pre-B cells but not mature B cells (Land et al., 1983; Schwartz et al., 1986; Stone et al., 1987; Overell et al., 1989). The REF cooperation assay has proven effective in the evaluation of candidate modulators of myc oncogenic potential. Using this assay it has been shown that the N-terminal TAD as well as the C-terminal region comprising the bHLHZip domain are of importance for the transforming activity of Myc (Sarid et al., 1987; Stone et al., 1987). The RatlA cells have been used in another in vitro assay for myc-transforming activity. These cells are immortalized but nontransformed and undergo neoplastic transformation upon introduction of a myc-expressing construct alone as shown by growth in soft agar and the ability to form tumors in nude mice (Stone et al., 1987). The regions of Myc that are important for transformation of RatlA differ slightly from the requirements for REF cotransformation (Stone et al., 1987). In addition, overexpression of myc acts synergistically with a61 oncogenes to cause transformation of certain fibroblasts and hematopoietic cells in vitro (Lug0 and Witte, 1989; Blackwood et al., 1994). Myc is believed to function downstream in the transformation pathway of v- Abl and Bcr-Abl. This notion is supported by the findings that dominant-negative forms of Myc block Bcr-Abl-mediated transformation and that coexpression of wildtype Myc could restore transformation to normal levels (Sawyers et al., 1992). In addition, single point mutations in the Src-homology 2 (SH2) domain or the Grb-2 binding site in the Bcr region impaired the transformation of fibroblasts by Bcr-Abl. Overexpression of Myc restored transformation by the Bcr-Abl SH2 mutant, indicating that Bcr-Abl activates at least two independent pathways for transformation (Afar et al., 1994). Myc overexpression similarly rescues an autophosphorylation mutant of the colonystimulating factor 1 (CSF-1) receptor for mitogenesis (Roussel et al., 1991). It is possible that Myc overexpression complements a pathway that is deficient in cells expressing either the autophosphorylation mutant of the CSF-1 receptor o r the SH2 mutant of Bcr-Abl. Interestingly, ectopic expression of cyclin D1, but not cyclin E, can also rescue the activity of the Bcr-Abl SH2 mutant in transformation, further supporting the concept of a close func-

Myc Proteins

161

tional interrelationship between c-Myc and cyclin D1 (Afar et af., 1995; see Section IV,F).

E. Analysis of c-Myc Transformation in Vitro I . T H E b H L H Z i p A N D INTERACTION WITH Max As mentioned previously, the bHLHZip domain is critical for Myc’s transforming activity. Furthermore, elegant studies in which mutant Myc and Max proteins were generated either by exchanging the HLHZip domains or by reciprocally modifying Zip dimerization specificities demonstrated that the oncogenic activity of Myc requires Max (Amati et af., 1993a). Likewise, Myc mutants either lacking the basic region or harboring the basic region of MyoD were unable to cooperate with bcr-abf in transformation, demonstrating that specific DNA binding is crucial for Myc transformation (Blackwood et af., 1994). In addition, a Myc mutant with deletion of aa 40-178 worked in a dominant-negative manner (Mukherjee et af., 1992). At higher levels, Max acts as a suppressor of Myc function and inhibits Myc transformation (Prendergast et af., 1992; Amati et af., 1993a; Cerni et af., 1995). The relative ratio of these proteins probably determines the resulting effects. While full-length Max suppresses the ability of Myc to transform REFS in cooperation with Ras, AMax enhances transformation (Makela et al., 1992). This was shown to be due to the lack of a full-length Zip (Vastrik et af., 1995b), but it may also suggest that there are regulatory factors binding to the C-terminus of Max. 2. T H E TRANSCRIPTIONAL ACTIVATION DOMAIN

The importance of the TAD in neoplastic transformation has been recognized through studies of Myc mutants as well as of comparisons with the related TAD of L-Myc. L-Myc is less potent than c-Myc in the REF cotransformation assay and its TAD was shown to have a reduced activity compared to the TAD of c-Myc (Birrer et af., 1988; Barrett et af., 1992). This might be explained by the fact that the glutamine-rich region as well as the region corresponding to amino acids 72-105 within the TAD of c-Myc are absent in L-Myc (Barrett et af., 1992). Furthermore, B-Myc was shown to squelch c-Myc-mediated transactivation as well as inhibit c-Myc-mediated transformation (Resar et al., 1993). Because B-Myc only contains a domain which is homologous to the amino terminus of c-Myc (Ingvarsson et al., 1988; Asker et af., 1995), these data suggest a correlation between Myc transactivation and transformation activity. However, no strict correlation between these two functions seems to exist

I62

M a r i e H e n r i k s s o n a n d B e r n h a r d Luscher

because there are mutants of c-Myc that are transactivating but fail to transform. For example, a study demonstrated that a protein fusing the aminoterminal 262 amino acids of c-Myc with the DNA-binding region of GAL4 [GAL4-Myc( 1-262)] could inhibit myclras cotransformation and that this inhibition was abolished when Myc Box I1 (amino acids 129-145) was deleted (Brough et al., 1995). However, mutations in or deletion of Myc Box I1 did not alter the transactivation potential of GAL4-Myc fusion proteins (Brough et al., 1995) or of c-Myc assayed on synthetic reporter genes (M. Henriksson, unpublished observation). Consequently, Myc Box I1 contains a domain that is essential for cell transformation but not for the transcriptional activity of c-Myc on synthetic promoters. However, it should be kept in mind that synthetic reporters may be misleading and d o not necessarily reflect the ability to activate cellular genes. As mentioned under Section IV an a-prothymosin reporter gene is no longer transactivated by Myc Box 11 mutants (M. Eilers, personal communication). Myc Box I1 seems to mediate the interaction with a nuclear factor, however, its nature is not defined at present (Brough et al., 1995). Two interesting possibilities are that it functions either as a coactivator connecting Myc to the basal transcription machinery o r as a DNA-binding protein further enhancing specificity.

3. SIGNIFICANCE OF THE PHOSPHORYLATION SITES WITHIN THE TAD The myc gene is frequently mutated in the second exon in a large number of BL-derived cell lines as well as in primary biopsies (Bhatia et al., 1993; Yano et al.. 1993; Albert et al., 1994). These clonal mutations are clustered in the region spanning amino acids 38-63 within the TAD. The same region is also affected by mutations in AIDS-related lymphomas (Bhatia et al., 1994; Clark et al., 1994), whereas no mutations have been found in other cell lines o r tumors without the Iglmyc translocation. Because the myc gene is already deregulated in these lymphomas as a result of the translocation, these mutations presumably affect critical regions that enhance Myc function, thereby providing a growth advantage to the tumor subclone. Within the affected region the muiations are clustered in the highly conserved Myc Box I implicating its biological significance. Two phosphorylation sites are localized in Myc Box I corresponding to Thr-58 and Ser-62 in human c-Myc. In fact, Thr-58 is the residue most frequently mutated, suggesting that phosphorylation at this site regulates Myc function. Interestingly, v-Myc proteins of MC29, MH2, and OK10, which are potent transformers, all contain a mutation at Thr-61, the equivalent amino acid to human Thr-58, to either Ala or Met (cited in Henriksson et al., 1993). A report has suggested that the mutations found in myc genes in BL affect the phosphorylation of the N-terminal sites (Hoang et al., 1995). As discussed under

Myc Proteins

163

Section III,E, mutant proteins still can bind the pl07/cyclin A/CDK complex but are no longer phosphorylated, implying that the mutations have a common effect. Mutations in myc genes from mouse plasmacytomas, which also contain Iglmyc rearrangements, seem to occur less frequently and the ones identified do not cluster around the N-terminal phosphorylation sites (Bhatia et al., 1993; Axelson et al., 1995). Several studies have shown that the transforming potential of Myc is altered when these two phosphorylation sites (Thr-58 and Ser-62) are mutated to nonphosphorylatable Ala (Henriksson et al., 1993; Pulverer et d., 1994). In the REF assay, Ala-58 enhanced, whereas Ala-62 reduced the transformation potential. Ala-58 also potentiated transformation in the RatlA assay, whereas Ala-62 behaved like wild type. It has also been suggested that these mutants affect Myc transactivation (Seth et al., 1991; Gupta et al., 1993; Albert et al., 1994). However, this has not been consistently observed (Henriksson et al., 1993; Lutterbach and Hann, 1994; M. Eilers, personal communication). One possibility of how these mutations could provide an advantage for tumor growth is by selectively inhibiting the ability of Myc to induce apoptosis without interfering with its growth-stimulatory function. The mutants described previously were tested for their ability to induce apoptosis in serum-starved fibroblasts. However, no significant differences were observed (Hoang et al., 1995; B. Amati, personal communication; M. Henriksson and B. Luscher, unpublished observation). At present the nature of the selective advantage provided by these mutations is unclear. 4. TRANSFORMATION BY P64Myc A N D P67Myc The translocations that occur in Burkitt lymphoma often disrupt the p67 initiation site (Hann et al., 1988) leading to the loss of p67Myc. Together with the induction of high levels of p67 in growth-inhibited cells (Hann et al., 1992), this suggests that p67Myc has a growth-inhibitory function. Perhaps when there is a disruption of p67 synthesis as a result of a genetic mutation or rearrangement, as in human BL and avian bursa1 lymphomas, specific cells lose a growth-inhibiting response to limiting nutrients which contributes to tumorigenicity. However, no difference in the transforming activity of p64 and p67Myc has been observed ilz vitro (Blackwood et al., 1994). It would be of interest to compare these two proteins in a transgenic model system.

5. EIA VERSUS Myc TRANSFORMATION Much interest was generated by the suggestion of structural similarities between c-Myc and E1A (Ralston and Bishop, 1983). It was established that both proteins can cooperate in transformation of primary embryo fibro-

I64

Marie Henriksson a n d Bernhard Luscher

blasts and BRK cells with an activated form of YUS (Land et al., 1983; Ruley, 1983). Subsequently, a detailed study of the transforming properties of E l A-Myc chimeras revealed functionally homologous domains (Ralston, 1991), suggesting that transformation by E1A and c-Myc may proceed through common targets. This idea is supported by the finding that Myc Box I1 interacts with a nuclear protein relevant for both E1A and Myc transformation (Brough et al., 1995). Previously it was shown that deletion of the transactivating domain in c-Myc, known to be required for transformation, resulted in a protein able to inhibit c-Myc/Ras but not ElA/Ras transformation. This was interpreted as evidence for independent modes of transformation of the two proteins (Mukherjee et al., 1992). However, these studies can be reconciled by the proposal that both common and distinct effectors of c-Myc and E1A transformation exist.

6. Myc AND THE CDK INHIBITOR p16 An important step in the progression from G1 into S is the hyperphosphorylation of Rb, which is thought to alter the activities of E2F family transcription factors (Wiman, 1993). This hyperphosphorylation is the result of the activation of cyclin D/CDK kinase complexes late in G1 (Sherr, 1994, 1995). p16ink4 specifically binds to and inhibits CDK4 and may thus regulate Rb phosphorylation. Indeed, transformed cells lacking functional Rb d o not require the activity of the cyclin D/CDK4 complexes and are hence insensitive to p16ink4. p16 appears to act as a tumor suppressor because the gene is frequently deleted in tumor cell lines and shows a high frequency of point mutations and small deletions in some tumor cell lines and primary tumors (see Hunter and Pines, 1994). Ectopic expression of p16ink4 suppresses cellular transformation of primary REFs by oncogenic Ha-ras and myc, but not by Ha-ras and E I A (Serrano et al., 1995). This suggests that whereas E1A can bind to Rb and thereby modulate E2F transcription factors, Myc is unable to directly impinge on Rb function and is therefore likely to control cell cycle progression upstream of Rb phosphorylation. This is compatible with the observed activation of cyclin D/CDK complexes in response to c-Myc as discussed above (see Section IV,G).

F. Influence of Mad Family Proteins on Transformation by Myc a n d Other Oncoproteins Several reports have demonstrated that Mad and M x i l repress c-Myc/Ras transformation of primary REFs in a dose-dependent manner (Lahoz er al., 1994; Cerni etal., 1995; Koskinen et al., 1995; Schreiber-Agus etal., 1995; Vastrik et al., 1995a), in agreement with their proposed role in inhibiting

Myc Proteins

165

Myc function. Both the number of foci and the severity of the malignant phenotype were reduced. Deletion of the basic region resulted in proteins that repressed transformation only mildly or not at all (Lahoz et al., 1994; Koskinen et al., 1995). Furthermore, a mutant lacking the Zip was incapable of inhibiting transformation, indicating that repression requires both DNA binding and interaction with Max (Koskinen et al., 1995). Mouse mxil expresses two mRNAs that arise through alternative RNA processing and that differ in their capacity to encode an amino-terminal extension of 36 residues, generating Mxi-SR and Mxi-WR (Schreiber-Agus et al., 1995; see Fig. 2). These proteins have different abilities to repress Myc-induced transformation. The presence of the amino-terminal segment correlates with a strong repressor activity a well as interaction with mSin3 proteins (Schreiber-Agus et al., 1995). A key role for this region is also suggested by its high conservation between M x i l proteins from different species as well as the initial observation that the homologous region in M a d binds mSin3 proteins (Ayer et al., 1995). The finding that Mxil-WR only possesses modest repressive potential despite having an intact bHLHZip is an indication against M x i l regulating Myc activity in a passive manner, either by titration of Max away from active Myc/Max or by the occupation of common binding sites by transactivation-incompetent Mxi 1/Max complexes as discussed under Section 111. Together, these findings provide a mechanistic basis for the antagonistic actions of M x i l and the other Mad family members on Myc activity that appears to be mediated in part through the recruitment of a putative transcriptional repressor. The analysis of Mad mutants also indicated that the N- and C-terminal homology regions are important for M a d function. Point mutations in the N-terminal region disrupting the putative a-helix or deletion of the C-terminal domain resulted in proteins with reduced repressing activity (Koskinen et al., 1995). A slightly more complex picture was observed in a second study in which deletions of either the N - or the C-terminal domain were still able to inhibit transformation by myc and ras (Cerni et al., 1995). However, whereas clones expressing wild-type Mad derived from the transformation assays grew very slowly, cells containing MadAN or MadAC did not elicit reduced growth. Furthermore, MadAN was no longer able to inhibit transformation by E1A and Ras (see below). In summary, these data indicate that in addition to the bHLHZip domain of Mad and Mxil, the other two homology regions appear indispensable for the full spectrum of functional activities. Besides inhibiting c-Myc/Ras transformation, Mad can also inhibit REF transformation by several other oncoproteins, including E l A, mutant p53, HPV16 E6 and E7 (Cerni et al., 1995), and cyclin D1 (T. Moroy, personal communication), in concert with Ras. This is an indication for a very broad repressing function of Mad, and together with its chromosomal location (see below) suggests that Mad may function as a tumor suppressor gene.

166

Marie Henriksson and Bernhard Luscher

The inhibitory effect of Mad on transformation cannot be abrogated by Bcl-2, suggesting that Mad affects the growth properties rather than the viability of cells (Koskinen et al., 1995). Furthermore, under conditions in which repression of Mad was most striking it was not possible to establish stable cell lines. Taken together, this suggests that overexpression of Mad is incompatible with Myc-Ras cotransformation as previously observed for Max. Recently, it was demonstrated that also Mad3 and Mad4 are efficient inhibitors of Myc/Ras cotransformation in REFS (Hurlin et af., 1995).

G. Chromosomal Localizations of max, mad, mxil, mad3, and mad4 Do These Genes Encode Tumor Suppressors? max has been mapped to mouse chromosome 12D and to chromosome 14q22-24 in humans (Gilladoga et al., 1992; Wagner et af., 1992; Table 1). This region of chromosome 14 is associated with a t(12;14)(q13-15;q2324) chromosomal translocation in uterine leiomyomas and recurrent deletions in some B cell chronic lymphocytic leukemias and malignant lymphomas [de1(14)(q22-24)] (Trent et al., 1989). Whether the function or the regulation of the max gene is altered as a result of these rearrangements is unknown. Because Max is essential for c-Myc function homozygous loss of this gene would be detrimental to cell growth. However, loss of one copy may influence the ratio between MycJMax and MaxJMax complexes in

Table I Chromosomal Localization of Myc Network Genes Chromosomal localization ~

Gene c-myc N-myc L-myc max mad mxi 1 mad3 mad4

Human 8q24 2~23-24 lp32 14q22-24 2p13 1 Oq25 5qa 4PU

(Ref.)

Mouse

~~~

(Ref.)

15D 12 4 12D 6 19D 13 5

Note. References: (1) Dalla-Favera et a/. (1982); (2) Crews et a/. (1982); (3) Schwab et a/. (1984); (4) Campbell et al. (1989); ( 5 ) Nau et al. (1985); (6) Gilladoga et al. (1992); (7) Edelhoff et a/. (1994); (8) Shapiro e t a / . (1994); (9) Wechsler et a/. (1994); (10) Hurlin e t a / . (19953). 9redicted synrenic regions based on the mouse chromosomal positions.

Myc Proteins

167

favor of the former which may result in growth promotion. This is based on the assumption that Max/Max homodimers antagonize Myc function, which they can upon overexpression, as discussed previously. However, whether this is their normal physiological role is far from clear. The analysis of the max locus in some of the previously mentioned chromosomal alterations should shed light on the potential role of Max as a tumor suppressor. In contrast to Max, the experimental evidence for Mad family proteins suggests that they might function as tumor suppressors. The mad gene has been mapped to human chromosome 2p13 and the mxil gene to chromosome 10q25 (Edelhoff et al., 1994; Shapiro et al., 1994). Both these regions are involved in translocations, deletions, or rearrangements in a number of human tumors implying that these genes might serve tumor suppressor functions (see Trent et al., 1989; Edelhoff et al., 1994; Shapiro et al., 1994; Eagle et al., 1995 and references therein). Support for this notion has come from a study demonstrating that the mxil gene is mutated or deleted in some prostate cancers (Eagle et al., 1995). Mad3 has been localized to the central region of mouse chromosome 13 and mad4 to the proximal region of mouse chromosome 5 (Hurlin et al., 1995a). The predicted human syntenic regions for mad3 and mad4, 5q and 4p, respectively, are candidate regions for the presence of genes associated with a number of different tumor types (see Hurlin et al., 1995a). Recently, it has also been shown that Mad can inhibit the growth as well as the tumorigenicity of human astrocytoma cells both in vitro and in a mouse xenograft model (Chen et al., 1995). Taken together with the data from in vitro studies in which Mad family proteins have been shown to suppress transformation and to inhibit cell growth by blocking S phase entry, these observations are consistent with a role of these proteins as tumor suppressors whose loss of function could serve as an important event in the development of some naturally occurring cancers. It will now be important to determine the role of the mad genes in some of the tumors with recurrent alterations of the corresponding regions; the expectation is that alterations at these loci will be found.

VIII. FUTURE ASPECTS The importance of c-Myc as growth regulator has been amply documented by its ability to drive cells into S phase, to inhibit differentiation, and also by its tumorigenic potential. It has been shown that both c-Myc and N-Myc are essential for early mouse development. It is likely that every cell in higher eukaryotes requires a Myc protein for correct growth. Because Max is the critical partner for c-Myc and probably also for N- and L-Myc, for all functions analyzed to date, it is satisfying to see that Max is also

168

Marie Henriksson and Bernhard Luscher

essential. In fact mux-l- embryos die even earlier than the ones deficient in c- or N-myc, probably owing to the fact that the function of all Myc proteins is impaired. With the identification of the Max partners, Mad, M x i l , Mad3, and Mad4, the Myc network has revealed two opposing sides, one promoting and one inhibiting growth. The question immediately arising is whether there are additional Max partners and proteins interacting with the M a d family to be identified which may offer even more complexity to the Myc network. This seems likely and it will be important to identify all members of the network to be able to analyze the interrelationships of the putative protein complexes. The different dimeric complexes within the network analyzed to date bind to the same, or very closely related, DNA sequences. It will therefore be necessary to determine if the same genes are regulated by these dimers. It seems possible that the group of genes regulated by individual protein complexes may not be identical. If this latter suggestion is correct it will be important to define the differences between these response elements. The identification of new target genes will also be important and will hopefully tell us how the Myc network proteins regulate cell growth. Furthermore, the regulation of the expression and the function of the Myc network genes and proteins, respectively, will have to be investigated. At present we know very little about how, for instance, the transcription or the splicing of the mud genes are regulated. Also the role of post-translational modification of the Myc network proteins is not well understood. It seems that, despite thousands of publications on Myc network genes and proteins, we have only scratched on the surface of what has to be learned to understand their biology. However, we envision that progress will be less hard to come by in the next few years in light of the recent findings described above.

ACKNOWLEDGMENTS We thank many colleagues for generously sharing unpublished data, for providing preprints

of manuscripts, and for sending reprints. We thank S. Burley for providing Fig. 3. We gratefully

acknowledge M. Cahill, D. Eick, J. Luscher-Firzlaff, H. Hermeking, L-G. Larsson, and A. Sommer for critical readings of the manuscript, S. Hilfenhaus, A. Menkel, and A. Sommer for help with the references, and A. Borchert for secretarial assistance. We also thank J. LiischerFirzlaff and S. Arsenian for their support and patience. Unpublished work from the authors’ laboratory was supported by grants from the Deutsche Krebshilfe (W38/92 Liil) and from the Deutsche Forschungsgemeinschaft (NO 120/6-3) to B.L. M.H. was supported in part by a grant from the Swedish Medical Research Council.

Myc Proteins

169

REFERENCES Abrahamsen, M . S., and Morris, D. R. (1990). Mol. Cell. Biol. 10, 5525-5528. Acton, D., Domen, J., Jacobs, H., Vlaar, M., Korsmeyer, S., and Berns, A. (1992). Curr. Topics. Microbiol. Immunol. 182, 293-298. Adams, J. M., Harris, A. W., Pinkert, C. A., Corcoran, L. M., Alexander, W. S., Cory, S., Palmiter, R. D., and Brinster, R. L. (1985). Nature 318, 533-538. Adnane, J., and Robbins, P. D. (1995). Oncogene 10,381-387. Afar, D.E.H., Goga, A., McLaughlin, J., Witte, 0. N., and Sawyers, C. L. (1994).Science 264, 424-426. Afar, D.E.H., McLaughlin, J., Sherr, C. J., Witte, 0. N., and Roussel, M. F. (1995). Proc. Natl. Acad. Sci. USA 92, 9540-9544. Akeson, R., and Bernards, R. (1990). Mol. Cell. Biol. 10, 2012-2016. Alber, T. (1992). Curr. Opin. Genet. Dev. 2, 205-210. Albert, T., Urlbauer, B., Kohlhuber, F., Hammersen, B., and Eick, D. (1994).Oncogene 9,759763. Alexander, W. S., Adams, J. M., and Cory, S. (1989a). Mol. Cell. Biol. 9, 67-73. Alexander, W. S., Bernard, O., Cory, S., and Adams, J. M. (1989b). Oncogene 4, 575-581. Alkema, M. J., van der Lugt, N.M.T., Bobeldijk, R. C., Berns, A., and van Lohuizen, M. (1995). Nature 374, 724-727. Alexandrova, N., Niklinski, J., Bliskovsky, V., Otterson, G. A., Blake, M., Kaye, F. J., and Zajak-Kaye, M. (1995). Mol. Cell. Biol. 15, 5188-5195. Alvarez, E., Northwood, 1. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991).1. Biol. Chem. 266, 15277-15285. Amati, B., Dalton, S., Brooks, M. W., Littlewood, T. D., Evan, G. I., and Land, H. (1992). Nature 359, 423-426. Amati, B., Brooks, M. W., Levy, N., Littlewood, T. D., Evan, G. I., and Land, H. (1993a). Cell 72, 233-245. Amati, B., Littlewood, T. D., Evan, G. I., and Land, H. (1993b). E M 5 0 /. 12, 5083-5087. Amin, C., Wagner, A. J., and Hay, N. (1993). Mol. Cell. Biol. 13, 383-390. Arany, Z., Newsome, D., Oldread, E., Livingston, D. M., and Eckner, R. (1995). Nature 374, 81-84. Arias, J., Alberts, A. S., Brindle, P., Claret, F. X., Smeal, T., Karin, M., Feramisco, J., and Montminy, M. (1994). Nature 370, 226-229. Armelin, H . A., Armelin, M.C.S., Kelly, K., Stewart, T., Leder, P., Cochran, B. H., and Stiles, C. D. (1984). Nature 310, 655-660. Asai, A., Miyagi, Y., Sugiyama, A., Kanemitsu, H., Obinata, M., Mishima, K., and Kuchino, Y. (1994). Oncogene 9,2345-2352. Asker, C., Magnusson, K. P., Piccoli, S. P., Anderson, K., Klein, G., Cole, M. D., and Wiman, K. G. (1995). Oncogene 11, 1963-1969. Askew, D., Ashum, R., Simmons, B., and Cleveland, J. (1991). Oncogene 6, 1915-1922. Asselin, C., and Marcu, K. B. (1989). Oncogene Res. 5, 67-72. Auvinen, M., Paasinen, A., Anderson, L. C., and Hollta, E. (1992). Nature 360, 355-358. Axelson, H., Henriksson, M., Wang, Y., Magnusson, K. P., and Klein, G. (1995). Eur. /. Cancer, in press. Ayer, D. E., and Eisenman, R. N. (1993). Genes Dev. 7, 2110-2119. Ayer, D. E., Kretzner, L., and Eisenman, R. N. (1993). Cell 72, 211-222. Ayer, D. E., Lawrence, Q. A., and Eisenman, R. N. (1995). Cell 80, 767-776. Bakhshi, A., Jensen, J. P., Goldman, P., Wright, J. J., McBride, 0. W., Epstein, A. L., and Korsmeyer, S. J. (1985). Cell 4, 899-906.

I70

Marie Henriksson and Bernhard Luscher

Barone, M. V., and Courtneidge, S. A. (1995).Nature 378, 509-512. Barrett, J., Birrer, M. J., Kato, G. J., Dosaka-Akita, H., and Dang, C. V. (1992).Mol. Cell. Biol. 12,3130-3137. Baxevanis, A. D., and Vinson, C. R. (1993). Curr. Opin. Genet. Deu. 3, 278-285. Beckmann, H., and Kadesch, T. (1991). Genes Deu. 5 , 1057-1066. Beckmann, H., Su, L.-K., and Kadesch, T. (1990). Genes Deu. 4, 167-179. Beer, D. G., Zweifel, K. A., Simpson, D. P., and Pitot, H. C. (1987).J.Cell. Physiol. 131,29-35. Beijersbergen, R. L., Hijmans, E. M., Zhu, L., and Bernards, R. (1994). E M B O J . 13, 40804086. Bello-Fernandez, C., Packham, G., and Cleveland, J. L. (1993). Proc. Natl. Acad. Sci. USA 90, 7804-7808. Benezra, R., Davis, R. L., Lockshon, D., Turner, D. L., and Weintraub, H. (1990).Cell 61,4959. Benvenisty, N., Leder, A., Kuo, A., and Leder, P. (1992). Genes Deu. 6, 2513-2523. Berberich, S. J., and Cole, M. D. (1992). Genes Dev. 6, 166-176. Berberich, S., Hyde-DeRuyscher, N., Espenshade, P., and Cole, M. (1992). Oncogene 7, 775779. Bernards, R., Dessain, S., and Weinberg, R. (1986). Cell 47, 667-674. Bernelot Moens, C., Auerbach, A. B., Conlon, R. A., Joyner, A. L., and Rossant, J. (1992). Genes Deu. 6, 691-704. Bhatia, K., Huppi, K., Spangle, G., Siwarski, D., lyer, R., and Magrath, 1. (1993). Nature Genet. 5, 56-61. Bhatia, K., Spangler, G., Gaidano, G., Hamdy, N., Dalla-Favera, R., and Magrath, I. (1994). Blood 84, 883-888. Bies, J., and Wolff, L. (1995). Cancer Res. 55, 501-504. Billaud, M., Isselbacher, K. J,, and Bernards, R. (1993).Proc. Natl. Acad. Sci. USA 90, 27392743. Birrer, M. J., Segal, S., DeGreve, J. S., Kaye, F., Sausville, E. A., and Minna, J. D. (1988). Mol. Cell. Biol. 8, 2668-2673. Bissonnette, R. P., Echeverri, F., Mahboubi, A., and Green, D. R. (1992). Nature 359, 552554. Bissonnette, R. P., McGahon, A., Mahboubi, A., and Green, D. R. (1994a).J. Exp. Med. 180, 2413-241 8. Bissonnette, R. P., Shi, Y.,Mohboubi, A., Glynn, J. M., and Green, D. R. (1994b). In “Apoptosis 11: The Molecular Basis of Apoptosis in Disease,” pp. 327-357. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Bister, K., and Jansen, K. W. (1986). Adu. Cancer Res. 47, 99-188. Blackwell, T. K., Kretzner, L., Blackwood, E. M., Eisenman, R. N., and Weintraub, H. ( I 990). Science 250, 1149-1151. Blackwell, T. K., Huang, J., Ma, A., Kretzner, L., Alt, F. W., Eisenman, R. N., and Weintraub, H. (1993). Mol. Cell. Biol. 13, 5216-5224. Blackwood, E. M., and Eisenman, R. N. (1991).Science 251, 1211-1217. Blackwood, E. M., Liischer, B., and Eisenman, R. N. (1992). Genes Deu. 6, 71-80. Blackwood, E. M., Lugo, T. G., Kretzner, L., King, M. W., Street, A. J., Witte, 0. N., and Eisenman, R. N. (1994). Mol. Biol. Cell 5, 597-609. Bodrug, S. E., Warner, B. J., Bath, M. L., Lindeman, G. J., Harris, A. W., and Adams, J. M. (1994). E M B O J. 13,2124-2130. Born, T. L., Frost, J. A., Schonthal, A., Prendergast, G. C., and Feramisco, J. R. (1994). Mol. Cell. Biol. 14, 5710-5718. Bousset, K., Henriksson, M., Luscher-Firzlaff, J. M., Litchfield, D. W., and Liischer, B. ( 1 993). Oncogene 8, 321 1-3220.

Myc Proteins

171

Bousset, K., Oelgeschlager, M., Henriksson, M., Schreek, S., Burkhardt, H., Litchfield, D. W., Luscher-Firzlaff, J. M., and Luscher, B. (1994). Cell. Mol. Biol. Res. 40, 501-511. Bousset, K., Oelgeschlager, M., and Luscher, B. (1995). Submitted for publication. Bowlin, T. L., McKnown, B. J., and Sunkara, P. S. (1986). Cell. Immunol. 98, 341-350. Brough, D. E., Hofmann, T. J., Ellwood, K. B., Townley, R. A., and Cole, M. D. (1995). Mol. Cell. Biol. 15, 1536-1544. Buckle, R. S., and Mechali, M. (1995). Oncogene 10, 1249-1255. Bunker, C. A., and Kingston, R. E. (1995). Nucleic Acids Res. 23, 269-276. Bustelo, X. R., Otero, A,, Gomez-Marquez, J., and Freire, M. (1991)./.Biol. Chem. 266,14431447. Campbell, G. R., Zimmerman, K., Blank, R. D., Alt, F. W., and D’Eustachio, P. (1989). Oncogene Res. 4, 47-54. Campisi, J., Gray, H. E., Pardee, A. B., Dean, M., and Sonnenshein, G. (1984). Cell 36, 241247. Carr, C. S., and Sharp, P. A. (1990). Mol. Cell. Biol. 10, 4384-4388. Cerni, C., Bousset, K., Seelos, C., Burkhardt, H., Henriksson, M., and Liischer, B. (1995). Oncogene 11, 587-596. Charron, J., Malynn, B. A., Fisher, P., Stewart, V., Jeannotte, L., Goff, S. P., Robertson, E. J., and Alt, F. W. (1992). Genes Dev. 6, 2248-2257. Chen, C., Biegalke, B. J., Eisenman, R. N., and Linial, M. L. (1989)./. Virol. 63,5092-5100. Chen, J., Willingham, T., Margraf, L. R., Schreiber-Agus, N., DePinho, R. A., and Nisen, P. D. (1995). Nature Med. 1, 638-643. Chou, T.-Y., Dang, C . V., and Hart, G. W. (1995a). Proc. Natl. Acad. Sci. USA 92, 44174421. Chou, T.-Y., Hart, G. W., and Dang, C. V. (1995b).1. B i d . Chem., 270, 18961-18965. Clark, H. M., Yano, T., Otsuki, T., Jaffe, E. S., Shibata, D., and Raffeld, M. (1994).Cancer Res. 54, 3383-3386. Cleary, M. L., Smith, S. D., and Sklar, J. (1986). Cell 47, 19-28. Cogliati, T., Dunn, B. K., Bar-Ner, M., Cultraro, C . M., and Segal, S. (1993). Oncogene 8, 1263-1268. Cohen, P. L., and Eisenberg, R. A. (1992). Trends Genet. 13, 427-428. Conteas, C. N., Mutchnik, M. G., Palmer, K. C., Weller, F. E., Luk, G. D., Naylor, P. H., Erdos, M. R., Goldstein, A. L., Panneerselvam, C., and Horecker, B. C. (1990). Proc. Natl. Acad. Sci. USA 87, 3269-3273. Coppola, J. A,, and Cole, M. D. (1986). Nature 320, 760-763. Cowley, B. D., Jr., Chadwick, L. J., Grantham, J. J., and Calvet, J. P. (1989)./.Biol. Chem. 264, 8389-8393. Craig, R. W., Buchan, H. L., Civin, C., and Kastan, M. B. (1993).Cell Growth Difer. 4,349357. Cress, W. D., and Triezenberg, S. J. (1991). Science 251, 87-90. Crews, S., Barth, R., Hood, L., Prehn, J., and Calame, K. (1982). Science 218, 1319-1321. Crouch, D. H., Lang, C., and Gillespie, D.A.F. (1990). Oncogene 5, 683-689. Crouch, D. H., Fischer, F., Clark, W., Jayaraman, P.-S., Goding, C. R., and Gillespie, D.A.F. (1993). Oncogene 8, 1849-1855. Cuypers, H. T., Selten, G., Quint, W., Zijlstra, M., Maandag, E. R., Boeiens, W., van Wezenbeek, P., Melief, C., and Berns, A. (1984). Cell 37, 141-150. Daksis, J., Lu, R. Y., Facchini, L. M., Marhin, W. W., and Penn, L.J.Z. (1994). Oncogene 9, 3 635-3 645. Dalla-Favera, R., Bregni, M., Ericson, J., Patterson, D., Gallo, R. C., and Croce, C. M. (1982). Proc. Natl. Acad. Sci. USA 79, 7824-7827. Dang, C. V., and Lee, W.M.F. (1988). Mol. Cell. Biol. 8, 4048-4054.

172

Marie Henriksson and Bernhard Liischer

Davis, A. C., Wims, M., Spotts, G. D., Hann, S. R., and Bradley, A. (1993).Genes Dev. 7, 671682. Davis, L. J., and Halazonetis, T. D. (1993). Oncogene 8, 125-132. Delgado, M. D., Lerga, A., Canelles, M., Gomez-Casares, M. T., and Leon, J. (1995). Oncogene 10, 1659-1665. DePinho, R. A,, Schreiber-Agus, N., and Ah, F. W. (1991). Adv. Cancer Res. 57, 1-46. Dikstein, R., Heffetz, D., Ben-Neriah, Y.,and Shaul, Y. (1992). Cell 69, 751-757. Dildrop, R., Ma, A., Zimmerman, K., Hsu, E., Tesfaye, A., DePinho, R., and Alt, F. W. (1989). E M B O J . 8 , 1121-1128. Dmitrovsky, E., Kuehl, W. M., Hollis, G. F., Kirsh, I. R., Bender, T. P., and Segal, S. (1986). Nature 322, 748-750. Domen, J., van der Lugt, N.M.T., Acton, D., Laird, P. W., Linders, K., and Berns, A. (1993a). J. Exp. M e d . 178, 1665-1673. Domen, J., van der Lugt, N.M.T., Laird, P. W., Saris, C.J.M., Clarke, A. R., Hooper, M. L., and Berns, A. (1993b). Blood 82, 1445-1452. Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J., Montgomery, J. C. A., Butel, J. S., and Bradley, A. (1992). Nature 356, 215-221. Dosil, M., Alvarez-Fernandez, L., and Gomez-Marquez, J. (1993). Exp. Cell. Res. 204, 94101. Dotto, P. G., Gilman, M., Maruyama, M., and Weinberg, R. (1986). E M B O J. 5, 2853-2857. Downs, K. M., Martin, G. R., and Bishop, J. M. (1989). Genes Dev. 3, 860-869. Dunn, B. K., Cogliati, T., Cultaro, C. M., Bar-Ner, M., and Segal, S. (1994). Cell Growth Differ. 5, 847-854. Eagle, L. R., Yin, X., Brothman, A. R., Williams, B. J., Atkin, N. B., and Prochownik, E. V. (1995). Nature Genet. 9, 249-255. Edelhoff, S., Ayer, D. E., Zervos, A. S., Steingrimsson, E., Jenkins, N. A., Copeland, N. G., Eisenman, R. N., Brent, R., and Disteche, C. M. (1994). Oncogene 9, 665-668. Eilers, M., Picard, D., Yamamoto, K. R., and Bishop, J. M. (1989). Nature 340, 66-68. Eilers, M., Schirm, S., and Bishop, J. M. (1991). E M B O J. 10, 133-141. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Parsons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinder, K. W., and Vogelstein, B. (1993). Cell 75, 817-825. Ellenberger, T. E., Brandt, C. J., Struhl, K., and Harrison, S. C. (1992). Cell 71, 1223-1237. Ellis, H. M., Spann, D. H., and Posakony, J. W. (1990). Cell 61, 27-38. Evan, G. I., Wyllie, A. H., Gilbert, C. S., Littlewood, T. D., Land, H., Brooks, M., Waters, C. M., Penn, L. Z., and Hancock, D. C. (1992). Cell 69, 119-128. Evans, C. A,, Owen-Lynch, P.J., Whetton, A. D., and Dive, C. (1993).J. Cancer Res. 53,17351738. Facchini, L. M., Chen, S., and Penn, L.J.Z. (1994). Cell Growth Differ. 5 , 637-646. Fanidi, A., Harrington, E. A., and Evan, G. 1. (1992).Nature 359, 554-556. Farell, P. J., Allan, G. J., Shanahan, F., Voudsen, K. H., and Crook, T. (1991). E M B O J. 10, 2879-2887. Ferre-D’Amare, A. R., Prendergast, G. C., Ziff, E. B., and Burley, S. K. (1993).Nature 363,3845. Ferre-D’Amare, A. R., Pognonec, P., Roeder, R. G., and Burley, S. K. (1994). EMBO J. 13, 180189. Field, J. K., and Spandidos, D. A. (1990). Anticancer Res. 10, 1-22. Fiers, W. (1991). FEBS Lett. 285, 199-212. Finke, J., Fritzen, R., Ternes, P., Trivedi, P., Bross, K. J., Lange, W., Mertelsmann, R., and Dolken, G. (1992). Blood 80, 459-469. Fisher, D. E. (1994). Cell 78, 539-542. Fisher, F., Jayaraman, P.-S., and Goding, C. R., (1991). Oncogene 6, 1099-1104.

Myc Proteins

173

Fisher, F., Crouch, D. H., Jayaraman, P.-S., Clark, W., Gillespie, D.A.F., and Goding, C. R. (1993). EMBO J. 12, 5075-5082. Freytag, S. (1988). Mol. Cell. Biol. 8, 1614-1624. Freytag, S. O., Dang, C. V., and Lee, W.M.F. (1990). Cell Growth Difer. 1, 339-343. Freytag, S. O., and Geddes, T. J. (1992).Science 256, 379-381. Gaidano, G., Ballerini, P., Gong, J. Z., Inghirami, G., Neri, A., Newcomb, E. W., Magrath, I. T., Knowles, D. M., and Dalla-Favera, R. (1991). Proc. Natl. Acad. Sci. USA 88, 54135417. Garell, J., and Modolell, J. (1990). Cell 61, 39-48. Gaubatz, S., Meichle, A., and Eilers, M. (1994). Mol. Cell. Biol. 14, 3853-3862. Gaubatz, S., Imhof, A., Dosch, R., Werner, O., Mitchell, P., Buettner, R., and Eilers, M. (1995). EMBO J. 14, 1508-1519. Gilladoga, A. D., Edelhoff, S., Blackwood, E. M., Eisenrnan, R. N., and Disteche, C. M. (1992). Oncogene 7, 1249-1251. Goff, S. P., Gilboa, E., Witte, 0. N., and Baltimore, D. (1980). Cell 22, 777-785. Gonda, T. J., Sheiness, D. K., and Bishop, J. M. (1982). Mol. Cell. Biol. 2, 617-624. Goodrich, D. W., and Lee, W.-H. (1992). Nature 360, 177-179. Goodrich, J. A., and Tjian, R. (1994). Curr. Opin. Cell. Biol. 6, 403-409. Goruppi, S., Gustincich, S., Brancolini, C., Lee, W.M.F., and Schneider, C. (1994).Oncogene9, 1537-1544. Graf, T., and Beug, H. (1978). Biochim. Biophys. Acta. 516, 269-299. Greenberg, M. E., and Ziff, E. B. (1984). Nature 311, 433-438. Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. C. (1994). Cancer Res. 54, 4855-4878. Gregor, P. D., Sawadago, M., and Roeder, R. G. (1990). Genes Deu. 4, 1730-1740. Gregory, C. D., Dive, C., Henderson, S., Smith, C. A., Williams, G. T., Gordon, J., and Rickinson, A. B. (1991). Nature 349, 612-614. Griep, A. E., and DeLuca, H. F. (1986). Proc. Natl. Acad. Sci. USA 83 5539-5543. Griep, A. E., and Westphal, H. (1988). Proc. Natl. Acad. Sci. USA 85, 6806-6810. Grignani, F., Lombardi, L., Inghirami, G., Sternas, L., Cechova, K., and Dalla-Favera, R. (1990). EMBO J. 9,3913-3922. Gu, W., Cechova, K., Tassi, V., and Dalla-Favera, R. (1993). Proc. Natl. Acad. Sci. USA 90, 2935-2939. Gu, W., Bhatia, K., Magrath, I. T., Dang, C. V., and Dalla-Favera, R. (1994).Science 264,251254. Gupta, S., Seth, A., and Davis, R. J. (1993). Proc. Natl. Acad. Sci. USA 90, 3216-3220. Gusse, M., Ghysdael, J., Evan, G., Soussi, T., and MCchali, M. (1989).Mol. Cell. Biol. 9,53955403. Halazonetis, T. D., and Kandil, A. N. (1991). Proc. Natl. Acad. Sci. U S A 88, 6162-6166. Hann, S. R., Thompson, C. B., and Eisenrnan, R. N. (1985). Nature 314, 366-369. Hann, S. R., King, M. W., Bentley, D. L., Anderson, C. W., and Eisenman, R. N. (1988). Cell 52, 185-195. Hann, S. R., Sloan-Brown, K., and Spotts, G. D. (1992). Genes Deu. 6, 1229-1240. Hann, S. R., Dixit, M., Sears, R. C., and Sealy, L. (1994). Genes Deu. 8, 2441-2452. Hanson, K. D., Shichiri, M., Follansbee, M. R., and Sedivy, J. M. (1994). Mol. Cell. Biol. 14, 5748-5755. Harrington, E. A,, Bennett, M. R., Fanidi, A., and Evan, G. I. (1994a). EMBO J. 13, 32863295. Harrington, E. A., Fanidi, A., and Evan, G. I. (1994b). Curr. Opin. Gen. Deu. 4, 120-129. Harris, A. W., Pinkert, C. A., Crawford, M., Langdon, W. Y., Brinster, R. L., and Adams, J. M. (1988).J. Exp. Med. 167, 353-371.

174

Marie Henriksson and Bernhard Liischer

Hateboer, G., Timmers, H.T.M., Rustgi, A. K., Billaud, M., van’t Veer, L. J., and Bernards, R. (1993). Proc. Natl. Acad. Sci. USA 90, 8489-8493. Haupt, Y., Alexander, W. S., Barri, G., Klinken, S. P., and A d a m , J. M. (1991).Cell 65, 753763. Hayashi, K., Makino, R., Kawamura, H., Arisawa, A., and Yoneda, K. (1987).Nucleic Acids Res. 15, 6419-6436. Heby, O., and Persson, L. (1990). Trends Biochern. Sci. 15, 153-158. Heikkila, R., Schwab, G., Wickstrom, E., Loke, S. L., Pluznik, D. H., Watt, R., and Neckers, L. M. (1987). Nature 328, 445-449. Henderson, S. M., Rose, M., Gregory, C., Croom-Carter, D., Wang, F., Longnecker, R., Kieff, E., and Rickinson, A. (1991). Cell 65, 1107-1115. Henderson, S., Huen, D., Rowe, M., Dawson, C., Johnson, G., and Rickinson, A. (1993).Proc. Natl. Acad. Sci. USA 90, 8479-8483. Henriksson, M., Bakardjiev, A,, Klein, G., and Liischer, B. (1993). Oncogene 8, 3199-3209. Hermeking, H., and Eick, D. (1994).Science 265, 2091-2093. Hermeking, H., Wolf, D. A., Kohlhuber, F., Dickmanns, A., Billaud, M., Fanning, E., and Eick, D. (1994). Proc. Natl. Acad. Sci. USA 91, 10412-10416. Hermeking, H., Funk, J. O., Reichert, M., Ellwart, J. W., and Eick, D. (1995). Oncogene 11, 1403-1409. Hibshoosh, H., Johnson, M., and Weinstein, 1. B. (1991).Oncogene 6, 739-743. Hinds, P. W., Dowdy, S. F., Eaton, E. N., Arnold, A., and Weinberg, R. A. (1994).Proc. Natl. Acad. Sci. USA 91, 709-713. Hirning, U., Schmid, P., Schulz, W. A., Rettenberger, G., and Hameister, H. (1991).Mech. Dev. 33, 119-126. Hirvonen, H., Makela, T. P., Sandberg, M., Kalimo, H., Vuorio, E., and Alitalo, K. (1990). Oncogene 5, 1787-1797. Hoang, A. T., Cohen, K. J., Barrett, J. F., Bergstrom, D. A., and Dang, C. V. (1994).Proc. Natl. Acad. Sci. USA 91, 6875-6879. Hoang, A. T., Lutterbach, B., Lewis, B. C., Yano, T., Chou, T.-Y., Barrett, J. F., Raffeld, M., Hann, S. R., and Dang, C. V. (1995). Mol. Cell. Biol. 15, 4031-4042. Holt, J. T., Fkdner, R. L., and Nienhuis, A. W. (1988). Mol. Cell. Biol. 8 , 963-973. Hopewell, R., and Ziff, E. B. (1995). Mol. Cell. Biol. 15, 3470-3478. Howes, K. A., Ranson, N., Papermaster, D. S., Lasudry, J. G. H., Albert, D. M., and Windle, J. J. (1994). Genes Dev. 8, 1300-1310. Hu, Y. F., Luscher, B., Admon, A., Mermod, N., and Tjian, R. (1990). Genes Dev. 4, 17411752. Hunter, T., and Pines, J. (1994). Cell 79, 573-582. Hurlin, P. J., Queva, C., Koskinen, P. J.. Steingrimsson, E., Ayer, D. E., Copeland, N. G., Jenkins, N. A., and Eisenman, R. N. (1995a). EMBO]., 14, 5646-5659. Hurlin, P. J., Foley, K. P.,Ayer, D. E., Eisenman, R. N., Hanahan, D., and Arbeit, J. M. (1995b). Oncogene, in press. Ingham, P. W., Howard, K. R., and Ish-Horowicz, D. (1985). Nature 318,439-445. Inghirami, G., Grignani, F., Sternas, L., Lombardi, L., Knowles, D., and Dalla-Favera, R. (1990). Science 250, 682-686. Ingvarsson, S. (1990). Cancer Biol. 1, 359-369. Ingvarsson, S., Asker, C., Axelson, H., Klein, G., and Sumegi, J. (1988). Mol. Cell. Biol. 8 , 3 168-3174. Jaattela, M., Wissing, D., Bauer, P. A., and Li, G. C. (1992). EMBO ]. 11, 3507-3512. Jacks, T., Remington, L., Williams, B. O., Schmitt, E. M., Halachmi, S., Bronson, R. T., and Weinberg, R. A. (1994). Curr. Biol. 4, 1-7. Jakobovits, A., Schwab, M., Bishop, J. M., and Martin, G. R. (1985). Nature 318, 188-191.

Myc Proteins

I75

Janicke, R. U., Lee, F.H.H., and Porter, A. G. (1994). Mol. Cell. Biol. 14, 5661-5670. Janknecht, R., Cahill, M. A., and Nordheim, A. (1995). Carcinogenesis 16, 443-450. Jansen-Diirr, P., Meichle, A., Steiner, P., Pagano, M., Finke, K., Botz, J., Wessbecher, J., Draetta, G., and Eilers, M. (1993). Proc. Natl. Acad. Sci. USA 90, 3685-3689. Johnson, P. F., Sterneck, E., and Williams, S. C. (1993).1. Nutr. Biochem. 4, 386-398. Kaddurah-Daouk, R., Green, J. M., Baldwin, A. S., Jr., and Kingston, R. E. (1987). Genes Dev. 1,347-357. Kalderon, D., Richardson, W. D., Markham, A. F., and Smith, A. E. (1984). Nature 311,3338. Karn, J., Watson, J. V., Lowe, A. D., Green, S. M., and Vedeckis, W. (1989). Oncogene 4,773787. Kato, G. J., Barrett, J., Villa-Garcia, M., and Dang, C. V. (1990). Mol. Cell. Biol. 10, 59145920. Kato, G. J., Lee, W. M. F., Chen, L., and Dang, C. V. (1992). Genes Dev. 6, 81-92. Kelly, K., Cochran, B. H., and Leder, P. (1983). Cell 35, 603-610. Kerkhoff, E., Bister, K., and Klempnauer, K.-H. (1991).Proc. Natl. Acad. Sci. USA 88,43234327. Kim, Y. H., Buchholz, M. A., Chrest, F. J., and Nordin, A. A. (1994).J. Immunol. 152,43284335. King, M. W., Roberts, J. M., and Eisenman, R. N. (1986). Mol. Cell. Biol. 6, 4499-4508. King, M. W., Blackwood, E. M., and Eisenman, R. N. (1993). Cell. Growth. Dev. 4, 85-92. Kingston, R. E., Baldwin, A. S., Jr., and Sharp, P. (1984). Nature 312, 280-282. Klambt, C., Knust, E., Tietze, K., and Campos-Ortega, J. A. (1989). EMBO J. 8, 203-210. Klefstrom, J., Vastrik, I., Saksela, E., Valle, J., Eilers, M., and Alitalo, K. (1994).E M B O J. 13, 5442-5450. Kohl, N. E., Kanda, N., Schreck, R. R., Bruns, G., Latt, S. A., Gilbert, F., and Alt, F. W. (1983). Cell 35, 359-367. Kohl, N. E., and Ruley, H. R. (1987). Oncogene 2, 41-48. Korsmeyer, S. J. (1995). Trends Genet. 11, 101-105. Koskinen, P. J., Vastrik, I., Makela, T. P., Eisenman, R. N., and Alitalo, K. (1994).Cell Growth Difer. 5, 313-320. Koskinen, P. J., Ayer, D. E., and Eisenman, R. N. (1995). Cell Growth Difer., 6 , 623-629. Kozopas, K. M., Yang, T., Buchan, H. L., Zhou, P., and Craig, R. W. (1993).Proc. Natl. Acad. Sci. USA 90, 35 16-3520. Kretzner, L., Blackwood, E. M., and Eisenman, R. N. (1992). Nature 359, 426-429. Krumm, A., Meulia, T., and Groudine, M. (1993). BioEssays 15, 659-665. Kumar, S., and Baglioni, C. (1991).J. Biol. Chem. 266, 20960-20964. Kurzrock, R., Gutterman, J. U., and Talpaz, M. (1988). N . Engl. J. Med. 319, 990-998. Kwok, R. P. S., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan, R. G., Roberts, S. G. E., Green, M. R., and Goodman, R. H. (1994). Nature 370, 223-226. Lachman, H. M., and Skoultchi, A. 1. (1984). Nature 310, 592-594. Lahoz, E. G., Xu, L., Schreiber-Agus, N., and DePinho, R. A. (1994). Proc. Natl. Acad. Sci. USA 91,5503-5507. Laird, P. W., van der Lugt, N. M. T., Clarke, A., Domen, J., Linders, K., McWhir, J., Berns, A., and Hooper, M. (1993). Nucleic Acids Res. 21, 4750-4755. Lalli, E., and Sassone-Corsi, P. (1994).J. Biol. Chem. 269, 17359-17362. Land, H., Parada, L. F., and Weinberg, R. A. (1983). Nature 304, 596-602. Landschulz, W. H., Johnson, P. F., and McKnight, S. L. (1988). Science 240, 1759-1764. Langdon, W. Y., Harris, A. W., Cory, S., and Adams, J. M. (1986). Cell 47, 11-18. Langdon, W. Y., Harris, A. W., and Cory, S. (1989). Oncogene Res. 4, 253-258. La Rocca, S. A., Crouch, D. H., and Gillespie, D. A. F. (1994). Oncogene 9, 3499-3508.

176

Marie Henriksson and Bernhard Luscher

Larrick, J. W., and Wright, S. C. (1990).FASEB J. 4, 3215-3223. Larsson, L.-G., Ivhed, I., Gidlund, M., Pettersson, U., Vennstrom, B., and Nilsson, K. (1988). Proc. Natl. Acad. Sci. USA 85, 2638-2642. Larsson, L.-G., Pettersson, M., Oberg, F., Nilsson, K., and Liischer, B. (1994). Oncogene 9, 1247- 1252. Lee, W. S., Kao, C., Bryant, G. O., Liu, X., and Berk, A. J. (1991). Cell 67, 365-376. Lernaitre, J.-M., Bocquet, S., Buckle, R., and Mechali, M. (1995). Mol. Cell. Biol. 15, 50545062. Li, L.-H., Nerlov, C., Prendergast, G., MacGregor, D., and Ziff, E. B. (1994). EMBO J. 13, 4070-4079. Lieberman, P. M., and Berk, A. J. (1990)./. Virol. 64, 2560-2568. Lin, Y.-S., Ha, I., Maldonado, E., Reinberg, D., and Green, M. R. (1991). Nature 353, 569571. Lindeman, G. J., Harris, A. W., Bath, M. L., Eisenman, R. N., and Adams, J. M. (1995). Oncogene 10, 1013-1017. Lovec, H., Grzeschiczek, A., Kowalski, M.-B., and Moroy, T. (1994a). EMBO /. 13, 34873495. Lovec, H., Sewing, A., Lucibello, F. C., Miiller, R., and Moroy, T. (1994b).Oncogene 9,323326. Lowe, S. W., Bodis, S., McClatchey, A., Remington, L., Ruley, H. E., Fisher, D. E., Housman, D. E., and Jacks, T. (1994).Science 266, 807-810. Lu, X., Park, S. H., Thompson, T. C., and Lane, D. P. (1992). Cell 70, 153-161. Lugo, T, G., and Witte, 0. N. (1989). Mol. Cell. Biol. 9, 1263-1270. Liischer B., and Eisenman, R. N . (1990). Genes Deu. 4, 2025-2035. Liischer, B., and Eisenman, R. N. (1992)./. Cell. Biol. 118, 775-784. Liischer, B., Kuenzel, E. A., Krebs, E. G., and Eisenman, R. N. (1989).EMBO J. 8,1111-1 119. Lutterbach, B., and Hann, S. R. (1994). Mol. Cell. Biol. 14, 5510-5522. Maheswaran, S., Lee, H., and Sonenshein, G. E. (1994).Mol. Cell. Biol. 14, 1147-1152. Makela, T. P., Koskinen, P. J., Vastrik, I., and Alitalo, K. (1992).Science 256, 373-377. Manrow, R. E., Sburlati, A. R., Hanover, J. A,, and Berger, S. I.. (1991).J. Biol. Chem. 266, 391 6-3924. Marcu, K. B., Bossone, S. A,, and Patel, A. J. (1992).Annu. Rev. Biochern. 61, 809-860. Marhin, W. W., Hei, Y.-J., Chen, S., Jiang, Z., Gallie, B. L., Phillips, R. A,, and Penn, L. J. 2. (1995). Oncogene, in press. Martel, C., Lallemand, D., and Crimisi, C. (1995). Oncogene 10, 2195-2205. McDonnell, T. J., Deane, N., Platt, F. M., Nunez, G., Jaeger, U., McKearn, J. P., and Korsmeyer, s. J., (1989). Cell 57, 79-88. McDonnell, T. J., Nunez, G., Platt, F. M., Hockenbery, D., London, L., McKearn, J. P., and Korsmeyer, S. J. (1990). Mol. Cell. Biol. 10, 1901-1907. McDonnell, T. J., and Korsmeyer, S. J. (1991). Nature 349, 254-256. McGahon, A., Bissonnette, R., Schmitt, M., Cotter, K. M., Green, D. R., and Cotter, T. G. (1994). Blood 83, 1179-1187. Milner, A. E., Johnson, G. D., and Gregory, C. D. (1992).Int. J. Cancer 52, 636-644. Milner, A. E., Grand, R. J. A., Waters, C. M., and Gregory, C. D. (1993).Oncogene 8,33853391. Miltenberger, R. J., Sukow, K. A., and Farnham, P. J. (1995). Mol. Cell. Biol. 15, 25272535. Min, S., and Taparowsky, E. J. (1992). Oncogene 7, 1531-1540. Miner, J. H., and Wold, B. J. (1991). Mol. Cell. Biol. 11, 2842-2851. Mitchell, P. J., and Tjian, R. (1989). Science 245, 371-378. Mori, M., Barnard, G. F., Staniumas, R. J., Jessup, J. M., Steele, G. D., Jr., and Chen, L. B. (1993). Oncogene 8,2821-2826.

Myc Proteins

177

Moroy, T., Fisher, P., Guidos, C., Ma, A., Zimmerman, K., Tesfaye, A., DePinho, R., Weissman, I., and Alt, F. W. (1990). E M B O J . 9, 3659-3666. Moroy, T., Grzeschiczek, A., Petzold, S., and Hartmann, K.-U., (1993). Proc. Natl. Acad. Sci. USA 90, 10734-10738. Moshier, J. A., Dosecu, J., Skunca, M., and Luk, G. D. (1993). Cancer Res. 53, 2618-2622. Mugrauer, G., Alt, F. W., and Ekblom, P. (1988).J. Cell. Biol. 107, 1325-1335. Mukherjee, B., Morgenbesser, S. D., and DePinho, R. A. (1992). Genes Dev. 6, 1480-1492. Murre, C., McCaw, P. S., and Baltimore, D. (1989). Cell 56, 777-783. Nath, P., Getzenberg, R., Beebe, D., Pallansch, L., and Zelenka, P. (1987). Exp. Cell Res. 169, 2 15-222. Nau, M. M., Brooks, B. J., Battey, J., Sausville, E., Gazdar, A. F., Kirsch, 1. R., McBride, 0. W., Bertness, V., Hollis, G. F., and Minna, J. D. (1985). Nature 318, 69-73. Nussenzweig, M. C., Schmidt, E. V., Shaw, A. C., Sinn, E., Campos-Torres, J., Mathey-Prevot, B., Pattengale, P. K., and Leder, P. (1988). Nature 336, 446-450. Oherg, F., Larsson, L.-G., Anton, R., and Nilsson, K. (1991). Proc. Natl. Acad. Sci. U S A 88, 5567-5571. Oelgeschlager, M., Janknecht, R., Krieg, J., and Liischer, B. (1995). Submitted for publication. Olson, E. N., (1990). Genes Deu. 4, 1454-1461. Onclercq, R., Gilardi, P., Lavenu, A., and Crimisi, C. (1988). J. Virol. 62,4533-4537. Onclercq, R., Bahinet, C., and Crimisi, C. (1989). Oncogene Res. 4, 293-302. Opipari, A. W., Jr., Hu, H. M., Yabkowitz, R., and Dixit, V. M. (1992). J. Biol. Chem. 267, 12424-12427. O’Shea, E. K., Klemm, J. D., Kim, P. S., and Alber, T. (1991). Science 254, 539-544. Overell, R. W., Weisser, K. E., Hess, B., Namen, A. E., and Grahstein, K. H. (1989). Oncogene 4, 1425-1432. Packham, G., and Cleveland, J. L. (1994). Mol. Cell. Biol. 14, 5741-5747. Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., Del Sal, G., Chau, V., Yew, P. R., Draetta, G. F., and Rolfe, M. (1995). Science 269, 682-685. Palmieri, S., Kahn, P., and Graf, T. (1983). E M B O J. 2, 2385-2389. Pan, H., and Griep, A. E. (1994). Genes Deu. 8, 1285-1299. Peltenburg, L. T. C., Dee, R., and Schrier, P. I. (1993). Nucleic Acids Res. 21, 1179-1185. Penn, L., Brooks, M., Laufer, E., Littlewood, T., Morgenstern, J., Evan, G., Lee, W., and Land, H. (1990a). Mol. Cell. Biol. 10, 4961-4966. Penn, L., Brooks, M., Laufer, E., and Land, H. (1990b). E M B O J . 9, 1113-1121. Pfeifer-Ohlsson, S., Goustin, A. S., Rydnert, J., Wahlstrom, T., Bjersing, L., Stehelin, D., and Ohlsson, R. (1984). Cell 38, 585-596. Pfeifer-Ohlsson, S., Rydnert, J., Goustin, A. S., Larsson, E., Betsholtz, C., and Ohlsson, R. (1985). Proc. Natl. Acad. Sci. USA 82, 5050-5054. Philipp, A., Schneider, A., Wstrik, I., Finke, K., Xiong, Y., Beach, D., Alitalo, K., and Eilers, M. (1994). Mol. Cell. Biol. 14, 4032-4043. Prendergast, G. C., and Cole, M. D. (1989). Mol. Cell. Biol. 9, 124-134. Prendergast, G. C., and Ziff, E. B. (1991). Science 251, 186-189. Prendergast, G. C., Diamond, L. E., Dahl, D., and Cole, M. D. (1990). Mol. Cell. Biol. 10, 1265-1269. Prendergast, G. C., Lawe, D., and Ziff, E. B. (1991). Cell 65, 395-407. Prendergast, G. C., Hopewell, R., Gorham, B. J., and Ziff, E. B. (1992). Genes Deu. 6, 24292439. Prochownik, E. W., Kokuwska, J., and Rogers, C. (1988). Mol. Cell. Biol. 8, 3683-3695. Pulverer, B. J., Fisher, C., Vousden, K., Littlewood, T., Evan, G., and Woodgett, J. R. (1994). Oncogene 9, 59-70. Rabbits, P. H., Watson, J. V., Lamond, A,, Forster, A., Stinson, M. A., Evans, G., Fischer, W., Atherton, E., Sheppard, R., and Rabbits, T. H. (1985). E M B O J. 4, 2009-2015.

178

Marie Henriksson and Bernhard Luscher

Raff, M. C., Barres, B. A., Burne, J. F., Coles, H. S., Ishizaki, Y., and Jacobson, M. D. (1993). Science 262, 695-700. Ralston, R. (1991).Nature 353, 866-868. Ralston, R., and Bishop, M. J. (1983). Nature 306, 803-806. Rao, L., Debbas, M., Sabbatini, P., Hockenberry, D., Korsmeyer, S., and White, E. (1992).Proc. Natl. Acad. Sci USA 89, 7742-7746. Reed, J. C. (1994).J. Cell Biol. 124, 1-6. Reed, J. C., Haldar, S., Croce, C. M., and Cuddy, M. P. (1990).Mol. Cell. Biol. 10,4370-4374. Reisman, D., Elkind, N. B., Roy, B., Beamon, J., and Rotter, V. (1993). Cell Growth Differ. 4, 57-65. Reitsma, P. H., Rothberg, P. G., Astrin, S. M., Trial, J., Bar-Shavit, Z., Hall, A., Teitelbaum, S. L., and Kahn, A. J. (1983). Nature 306, 492-494. Resar, L. M. S., Dolde, C., Barrett, J. F., and Dang, C. V. (1993). Mol. Cell. Biol. 13, 11301136. Reynolds, J. E., Yang, T., Qian, L., Jenkinson, J. D., Zhou, P., Eastman, A,, and Craig, R. W. (1994). Cancer Res. 54, 6348-6352. Ribon, V., Leff, T., and Saltiel, A. R. (1994). Mol. Cell. Neurosci. 5 , 277-282. Richardson, W. D., Roberts, B. L., and Smith, A. E. (1986). Cell 44, 77-85. Riechmann, V., van Criichten, I., and Sablitzky, F. (1994). Nucleic Acids Res. 22, 749-755. Riser, R. (1982). Yiochim. Biophys. Act. 651, 213-244. Ron, D., and Habener, J. F. (1992). Genes Dev. 6, 439-453. Ronen, D., Rotter, V., and Reisman, D. (1991). Proc. Natl. Acad. Sci. USA 88, 4128-4132. Rosenbaum, H., Webb, E., Adarns, J., Cory, S., and Harris, A. (1989).E M B O J . 8,749-755. Rosenbaum, H., Harris, A. W., Bath, M. L., McNeall, J., Webb, E., Adams, J. M., and Cory, S. (1990). E M B O J . 9, 897-905. Rosenberg, N., and Witte, 0. N. (1988).Adv. Virus Res. 35, 39-81. Rosenwald, 1. B., Rhoads, D. B., Callanan, L. D., Isselbacher, K. J., and Schmidt, E. V. (1993a). Proc. Natl. Acad. Sci. USA 90, 6175-6178. Rosenwald, I. B., Lazaris-Karatzas, A., Sonenberg, N., and Schmidt, E. V. (199313). Mol. Cell. Biol. 13, 7358-7363. Roussel, M. F., Shurtleff, S. A., Downing, J. R., and Sherr, C. J. (1990).I’roc. Nut/. Acad. Sci. U S A 87,6738-6742. Roussel, M. F., Cleveland, J. L., Shurtleff, S. A., and Sherr, C. J. (1991).Nature 353,361-363. Roussel, M. F., Davis, J. N., Cleveland, J. L., Ghysdael, J., and Hiebert, S. W. (1994).Oncogene 9,405-415. Roussel, M. F., Theodoras, A. M., Pagano, M., and Sherr, C. J. (1995).Proc. Natl. Acad. Sci. USA 92, 6837-6841. Roy, A. I.., Carruthers, C., Gutjahr, T., and Roeder, R. G. (1993). Nature 365, 359-361. Roy, B., Beamon, J., Balint, E., and Reisman, D. (1994).M o l . Cell. Biol. 14, 7805-7815. Ruley, H. E. (1983).Nature 304, 602-606. Rustgi, A. K., Dyson, N., and Bernards, R. (1991). Nature 352, 541-544. Sarid, J., Halazonetis, T. D., Murphy, W., and Leder, P. (1987).Proc. Natl. Acad. Sci. USA 84, 170-1 73. Saris, C. J. M., Domen, J., and Berns, A. (1991). E M B O J . 10, 655-664. Sasai, Y., Kageyama, R., Tagawa, Y., Shigemoto, R., and Nakanishi, S. (1992). Genes Dev. 6, 2620-2634. Sawai, S., Shimono, A., Hanaoka, K., and Kondoh, H. (1991). New Biologist 3, 8 6 1 4 6 9 . Sawyers, C. L., Callahan, W., and Witte, 0. N. (1992).Cell 70, 901-910. Sburlati, A. R., Manrow, R. E., and Berger, S. L. (1991).Proc. Natl. Acad. Sci. USA 88, 253257. Schmid, P., Schulz, W. A., and Hameister, H. (1989).Science 243, 226-229.

Myc Proteins

179

Schreiber-Agus, N., Homer, J., Torres, R., Chiu, F.-C., and DePinho, R. A. (1993).Mol. Cell. Biol. 13, 2765-2775. Schreiber-Agus, N., Chin, L., Chen, K., Torres, R., Rao, G., Guida, P., Skoultchi, A. I., and DePinho, R. A. (1995). Cell 80, 777-786. Schwab, M., Alitalo, K., Klempnauer, K. H., Varmus, H. E., Bishop, J. M., Gilbert, G., Brodeur, M., Goldstein, M., and Trent, J. (1983). Nature 305, 245-248. Schwab, M., Varmus, H. E., Bishop, J. M., Grzeschik, K. H., Naylor, S. L., Sakaguchi, A. Y., Brodeur, G., and Trant, J. (1984). Nature 308, 288-291. Schwartz, R. C., Stanton, L. W., Riley, S. C., Marcu, K. B., and Witte, 0. N. (1986). Mol. Cell. Biol. 6, 3221-3231. Schwartzberg, P. L., Stall, A. M., Hardin, J. D., Bowdish, K. S., Humaran, T., Boast, S., Harbison, M. L., Robertson, E. J., and Goff, S. P. (1991). Cell 65, 1165-1175. Seipel, K., Geogriev, O., and Schaffner, W. (1994). Biol. Chem. Hoppe-Seyler 375, 463-470. Selivanova, G., and Wiman, K. G. (1995). Adv. Cancer Res. 66, 143-180. Selten, G., Cuypers, H. T., and Berns, A. (1985).E M B O J. 4, 1793-1798. Selvakumaran, M., Lin, H.-K., Sjin, R. T. T., Reed, J. C., Libermann, D. A., and Hoffman, B. (1994). Mol. Cell. Biol. 14, 2352-2360. Semsei, I., Ma, S., and Cutler, R. G. (1989). Oncogene 4, 465-470. Serrano, M., Lahoz, E. G., DePinho, R., Beach, D., and Bar-Sagi, D. (1995).Science 267,249252. Seth, A., Alvarez, E., Gupta, S., and Davis, R. J. (1991).J. Biol. Chem. 266, 23521-23524. Seto, M., Jaeger, U., Hockett, R. D., Graninger, W., Bennett, S., Goldman, P., and Korsrneyer, S. J. (1988).E M B O J. 2, 123-131. Shan, B., and Lee, W.-H. (1994). Mol. Cell. Biol. 14, 8166-8173. Shapiro, D. N., Valentine, V., Eagle, L., Yin, X., Morris, S. W., and Prochownik, E. V. (1994). Genomics 23,282-285. Sheiness, D., and Bishop, J. M. (1979).J. Virol. 31, 514-521. Sheiness, D., Fanshier, L., and Bishop, J. M. (1978).J. Virol. 28, 600-610. Sherr, C. J. (1994). Cell 79, 551-555. Sherr, C. J. (1995). TZBS 20, 187-190. Shi, Y., Glynn, J. M., Guilbert, L. J., Cotter, T. G., Bissonnette, R. P., and Green, D. R. (1992). Science 257, 212-214. Shichiri, M., Hanson, K. D., and Sedivy, J. M. (1993). Cell Growth Differ. 4, 93-104. Shindo, H., Tani, E., Matsumoto, T., Hashimoto, T., and Furuyama, J. (1993). Acta Neuropathol. 86, 345-352. Shrivastava, A., and Calame, K. (1994).Nucleic Acids Res. 22, 5151-5155. Shrivastava, A., Saleque, S., Kalpana, G . V., Artandi, S., Goff, S . P., and Calame, K. (1993). Science 262, 1889-1892. Sinn, E., Muller, W., Pattengale, P.,Tepler, I., Wallace, R., and Leder, P. (1987).Cell 49, 465475. Smarda, J., and Lipsick, J. S. (1994). Oncogene 9, 237-245. Smith, M. J., Charron-Prochownik, D. C., and Prochownik, E. V. (1990).Mol. Cell. Biol. 10, 5333-5339. Smith, M. R., Al-Katib, A., Mohammad, R., Silverman, A., Szabo, P., Khilnani, S., Kohler, W., Nath, R., and Mutchnik, M. G. (1993). Blood 82, 1127-1 132. Sollenberger, K. G., Kao, T.-L., and Taparowsky, J. (1994). Oncogene 9, 661-664. Solomon, D.L.C., Amati, B., and Land, H. (1993). Nucleic Acids Res. 21, 5372-5376. Solomon, D., Philipp, A., Land, H., and Eilers, M. (1995). Oncogene 11, 1893-1897. Sorrentino, V., Drozdoff, V., McKinney, M. D., Zeitz, L., and Fleissner, E. (1986).Proc. Natl. Acad. Sci. USA 83, 8167-8171. Spencer, C. A., and Groudine, M. (1991). Adv. Cancer Res. 56, 1-48.

I80

Marie Henriksson and Bernhard Luscher

St. Arnand, R., Nepveu, A., Marcu, K. B., and McBurney, M. W. (1988). Oncogene 3, 553559. Stanton, B. R., Perkins, A. S., Tessarollo, L., Sassoon, D. A,, and Parada, L. F. (1992). Genes Dev. 6, 2235-2247. Stanton, L. W., Fahrlander, P. D., Tesser, P. M., and Marcu, K. B. (1984). Nature 310, 423425. Steiner, P., Philipp, A., Lukas, J., Godden-Kent, D., Pagano, M., Mittnacht, S., Bartek, J., and Eilers, M. (1995). EMBO I., 14, 4814-4826. Steller, H. (1995). Science 267, 1445-1449. Stern, D. F., Roberts, A. B., Roche, N. S., Sporn, M. B., and Weinberg, R. A. (1986). Mol. Cell. Biol. 6, 870-877. Stewart, T. A., Pattengale, P. K., and Leder, P. (1984). Cell 38, 627-637. Stone, J., de Lange, T., Ramsay, G., Jakobovits, E., Bishop, J. M., Varmus, H., and Lee, W. (1987). Mol. Cell. Biol. 7, 1697-1709. Strasser, A., Harris, A. W., Vaux, D. L., Webb, E., Bath, M. L., Adams, J. M., and Cory, S . (1990a). Curr. Topics, Microbiol. Immunol. 166, 175-181. Strasser, A., Harris, A. W., Bath, M. L., and Cory, S. (1990b). Nature 348, 331-333. Strasser, A., Whittingham, S., Vaux, D. L., Bath, M. L., Adams, J. M., Cory, S., and Harris, A. W. (1991). Proc. Natl. Acad. Sci. USA 88, 8661-8665. Strasser, A., Harris, A. W., and Cory, S. (1993). Oncogene 8, 1-9. Street, A. J., Blackwood, E., Luscher, B., and Eisenman, R. N. (1990). Curr. Topics. Microbiol. Imrnunol. 166,251-258. Stringer, K. F., Ingles, C . J., and Greenblatt, J. (1990). Nature 345, 783-786. Sugiyama, A., Kume, A., Nemoto, K., Lee, S . Y., Asami, Y., Nemoto, F., Nishimura, S., and Kuchino, Y. (1989). Proc. Natl. Acad. Sci. USA 86, 9144-9148. Taylor, M. V., Gusse, M., Evan, G. I., Dathan, N., and Mechali, M. (1986).EMBO ].5,35633570. te Riele, H., Maandag, E. R., Clarke, A., Hooper, M., and Berns, A. (1990). Nature 348,649651. Thompson, C. B., (1995). Science 267, 1456-1462. Thompson, C. B., Challoner, P. B., Neiman, P. E., and Groudine, M. (1985). Nature 314,363366. Thompson, N. L., Mead, J. E., Braun, L., Goyette, M., Shank, P. R., and Fausto, N. (1986). Cancer Res. 46, 31 11-3117. Thulasi, R., Harbour, D. V., and Thompson, E. B. (1993).J. Biol. Chem. 268, 18306-18312. Tietze, K., Oellers, N., and Knust, E. (1992). Proc. Natl. Acad. Sci. USA 89, 6152-6156. Tonissen, K. F., and Krieg, P. A. (1994). Oncogene 9, 33-38. Trent, J. M., Kaneko, Y., and Mitelman, F. (1989). Cytogenet. Cell. Genet. 51, 533-562. Triezenberg, S. J. (1995). Curr. Opin. Genet. Deu. 5 , 190-196. Tsujimoto, Y.,Gorham, J., Cossman, J., Jaffe, E., and Croce, C. M. (1985). Science 229, 13901393. Tsujimoto, Y., Bashir, M. M., Gwol, I., Cossman, J., Jaffe, E., and Croce, C. M. (1987). Proc. Natl. Acad. Sci. USA 84, 1329-1331. Tybulewicz, V. L. J., Crawford, C. E., Jackson, P. K., Bronson, R. T., and Mulligan, R. C. (1991). Cell 65, 1153-1163. van Beneden, R. J., Watson, D. K., Chen, T. T., Lautenberger, J. A., and Papas, T. S. (1986). Proc. Natl. Acad. Sci. USA 83,3698-3702. van der Lugt, N. M. T., Domen, J., Linders, K., van Roon, M., Maandag, E. R., te Riele, H., van der Valk, M., Deschamps, J., Sofroniew, M., van Lohuizen, M., and Berns, A. (1994). Genes Deu. 8. 757-769.

Myc Proteins

181

van de Lugt, N. M., Domen, J., Verhoeven, E., Linders, K., van der Gulden, H., Allen, J., and Berns, A. (1995). E M B O ] . 14, 2536-2544. van Lohuizen, M., and Berns, A. (1990). Biochin?. Biophys. Acta. 1032, 213-235. van Lohuizen, M., Verbeek, S., Krimpenfort, P., Domen, J., Saris, C., Radaszkiewicz, T., and Berns, A. (1989). Cell 56, 673-682. van Lohuizen, M., Verbeek, S., Scheijen, B., Wientjens, E., van der Gulden, H., and Berns, A. (1991). Cell 65, 737-752. van’t Veer, L. J., Beijersbergen, R. L., and Bernards, R. (1993). E M B O J . 12, 195-200. Vastrik, I., Kaipainen, A., Penttila, T.-L., Lymboussakis, A., Alitalo, R., Parvinen, M., and Alitalo, K. (1995a). I. Cell Biol. 128, 1197-1208. Vastrik, I., Makela, T. P., Koskinen, P. J., and Alitalo, K. (1995b). Oncogene 11, 533-560. Vaux, D. L., and Weissman, 1. L. (1993). Mol. Cell. Biol. 13, 7000-7005. Vaux, D. L., Cory, S., and Adams, J. M. (1988). Nature 335, 440-442. Vaux, D. L., Haecker, G., and Strasser, A. (1994). Cell 76, 777-779. Veis, D. J., Sorenson, C. M., Shutter, J. R., and Korsmeyer, S. J. (1993). Cell 75, 229-240. Vennstrom, B., Sheiness, D., Zabielski, J., and Bishop, J. M. (1982)./. Virol. 42, 773-779. Verbeek, S., van Lohuizen, M., van der Valk, M., Domen, J., Kraal, G., and Berns, A. (1991). Mol. Cell. Biol. 11, 1176-1179. Versteeg, R., Noordermeer, I., Kruse-Wolters, M., Ruiter, D., and Schrier, P. (1988). EMBO /. 7, 1023-1029. Vogelstein, B., and Kinzler, K. W. (1992). Cell 70, 523-526. Wagner, A. J., Le Beau, M. M., Diaz, M. O., and Hay, N. (1992). Proc. Natl. Acad. Sci. USA 89, 3111-3115. Wagner, A. J., Meyers, C., Laimins, L. A., and Hay, N. (1993a). Cell Growth Differ. 4, 879883. Wagner, A. J., Small, M. B., and Hay, N. (1993b). Mol. Cell. Biol. 13, 2432-2440. Wagner, A. J., Kokontis, J. M., and Hay, N. (1994). Genes Deu. 8, 2817-2830. Wakamatsu, Y., Watanabe, Y., Shimono, A., and Kondoh, H. (1993). Neuron 10, 1-9. Walker, C. W., Boom, J. D. G., and Marsh, A. G. (1992). Oncogene 7,2007-2012. Wang, J. Y. J. (1993). Curr. Biol. 3, 35-43. Wang, H., and Stillman, D. J. (1990). Proc. Natl. Acad. Sci. USA 87, 9761-9765. Wang, H., and Stillman, D. J. (1993). Mol. Cell. Biol. 13, 1805-1814. Watanabe-Fukunaga, R., Brannan, C . I., Copeland, N. G . , Jenkins, N. A., and Nagata, S. (1992). Nature 365, 314-317. Waters, C. M., Littlewood, T. D., Hancock, D. C., Moore, J. P., and Evan, G. I. (1991). Oncogene 6, 797-805. Wechsler, D. S., Hawkins, A. L., Li, X., Wang, J. E., Griffin, C. A., and Dang, C. V. (1994). Genomics 21, 669. Weissinger, E. M., Mischak, H., Goodnight, J., Davidson, W. F., and Mushinski, J. F. (1993). Mol. Cell. Biol. 13, 2578-2585. Wenzel, A., Cziepluch, C., Hamann, U., Schiirmann, J., and Schwab, M. (1991). E M B O ] . 10, 3703-3712. Wiman, K. G . (1993). FASEB]. 7, 841-845. Wiman, K. G., Magnusson, K. P., Ramqvist, T., and Klein, G. (1991). Oncogene 6, 16331639. Wong, G. H. W., Elwell, J. H., Oberley, L. W., and Goeddel, D. V. (1989). Cell 58, 923-931. Wurm, F., Gwinn, K., and Kingston, R. (1986). Proc. Natl. Acad. Sci. USA 83, 5414-5418. Wyllie, A. H. (1985). Anticancer Research 5, 131-136. Wyllie, A. H., Rose, K. A,, Morris, R. G., Steel, C. M., Foster, E., and Spandidos, D. A. (1987). BY.I. Cancer 56, 251-259.

I82

Marie Henriksson and Bernhard Luscher

Xiao, H., Pearson, A., Coulornbe, B., Truat, R., Zhang, S., Regier, J. L., Triezenberg, S. J., Reinberg, D., Flores, O., Ingles, C. J., and Greenblatt, J. (1994). Mol. Cell. Biol. 14, 70137024. Yang, B.-S., Geddes, T. J., Pogulis, R. J., de Crornbrugghe, B., and Freytag, S. 0. (1991). Mol. Cell. Biol. 11, 2291-229s. Yang, B.-S., Gilbert, J. D., and Freytag, S. 0. (1993). Mol. Cell. Biol. 13, 3093-3102. Yano, T., Sander, C. A., Clark, H. M., Dolezal, M. V., Jaffe, E. S . , and Raffeld, M. (1993). Oncogene 8 , 2741-2748. Yunis, J. J., Frizzera, G., Oken, M. M., McKenna, J., Theologides, A., and Arnesen, M. (1987). N . Engl. 1. Med. 316, 79-84. Zervos, A. S., Gyuris, J., and Brent, R. (1993). Cell 72, 223-232. Zimmerrnan, K. A., Yancopoulos, G. D., Collurn, R. G., Smith, R. K., Kohl, N. E., Denis, K. A., Nau, M. M., Witte, 0. N., Toran-Allerand, D., Gee, C . E., Minna, J. D., and Alt, F. W. (1986). Nature 319, 780-783.