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The Myc transcription factor is a potent inducer of proliferation and is required for Wnt/beta-catenin signaling in intestinal epithelium. Since deregulation of the Wnt/beta-catenin pathway is a prerequisite for nonhereditary intestinal tumorigenesis, we asked whether activation of Myc recapitulates the tumorigenic changes that are driven by constitutive Wnt/beta-catenin pathway signaling following adenomatous polyposis coli (APC) inactivation. Using mice in which expression of MycER(TAM), a reversibly switchable form of Myc, is expressed transgenically in intestinal epithelium, we define the acute changes that follow Myc activation as well as subsequent deactivation. Myc activation reversibly recapitulates many, but not all, aspects of APC inactivation, including increased proliferation and apoptosis and loss of goblet cells. However, whereas APC inactivation induces redistribution of Paneth cells, direct Myc activation triggers their rapid attrition. Moreover, direct Myc activation engages the ARF/p53/p21(cip1) tumor suppressor pathway, whereas deregulation of Wnt/beta-catenin signaling does not. These observations illustrate key differences in oncogenic impact in intestinal epithelium of direct Myc activation and indirect Myc activation via the Wnt/beta-catenin pathway. Furthermore, the in situ dedifferentiation of mature goblet cells that Myc induces indicates a novel cross talk between the Wnt/beta-catenin and Notch signaling pathways.

Myc is a transcription factor with pleiotropic effects on tumorigenesis which are likely to be mediated by its target genes. A known Myc transcriptional target is the catalytic subunit of telomerase, Tert. However, the contribution of Tert activation to Myc-induced tumorigenesis in vivo remains unknown. In this study, we addressed the role of telomerase in Myc-induced skin papillomatosis by using compound mice with a switchable Myc gene, Inv-MycERTAM mice, in combination with either telomerase deficiency (Terc−/−) or telomerase overexpression (K5-mTert) in the skin. We first demonstrated that Myc activates telomerase in the skin. With Inv-MycERTAM × Terc−/− mice, we further showed that this telomerase activation is partially required to elicit a full hyperplastic Myc-induced response. The presence of critically short telomeres in late-generation Inv-MycERTAM × Terc−/− mice further reduced the skin lesion induced by Myc. On the other hand, telomerase overexpression in the skin of K5-mTert mice augments Myc-induced hyperplasia in the absence of changes in telomere length, suggesting a direct role of telomerase in the Myc protumorigenic response. Taken together, these results highlight telomerase as a mediator of Myc-induced papillomatosis and suggest telomerase as a putative therapeutic target for Myc-dependent lesions.

ocytes, culminating in papillomatosis. Transcription of the Id2 gene is regulated by Myc family proteins. Moreover, Id2 is implicated as a pivotal determinant of cell fate in multiple lineages and has a demonstrated role in mediating Myc-dependent cell proliferation in vitro through its interaction with retinoblastoma protein. Using Id2 nullizygous mice, we assessed in vivo the requirement for Id2 in mediating Myc-induced papilloma formation in skin. We show that absence of Id2 has no discernible impact on any measurable attribute of Myc function or on the timing or extent of eventual tumor formation. Thus, our data argue against any essential role for Id2 in mediating Myc action in vivo. Deregulated expression of myc family oncogenes occurs in a wide range of human cancers and is often associated with aggressive, poorly differentiated tumors (28). As members of the bHLH-Zip transcription factor family, Myc proteins are thought to exert their biological effects by modulating, either positively or negatively, expression of downstream target genes (24). Indeed, recent advances in expression array and serial analysis of gene expression technologies have indicated that Myc proteins regulate a bewilderingly diverse variety of genes which are variously implicated in almost every aspect of cellular activity, including proliferation and differentiation, growth and apoptosis, metabolism, intracellular transport and organization, and interactions with the somatic cell environment (6, 10, 27, 31, 41). The breadth of potential Myc target genes, together with the highly contingent nature of the pathways and processes they oversee, makes it a great challenge to determine which attributes of Myc proteins are most critical for their tumorigenic properties. The Id2 gene was recently identified as a direct transcriptional target of both N-Myc and c-Myc (23). Using a conditional form of N-Myc (N-MycER), in which N-Myc activity is rendered dependent on the presence of the ligand 4-hydroxytamoxifen (4-OHT) by fusion with the estrogen receptor ligand binding domain (25), activation of N-Myc was demonstrated to rapidly induce expression of Id2 at both mRNA and protein levels. Induction of Id2 by N-Myc (and by c-Myc) is dependent on the presence of two Myc consensus E boxes located within the Id2 5 regulatory region. Consistent with this, chromatin immunoprecipitation analysis has indicated binding of c-Myc/ Max heterodimers to the Id2 promoter (23). Moreover, trans

c-Myc promotes apoptosis by destabilizing mitochondrial integrity, leading to the release of proapoptotic effectors including holocytochrome c. Candidate mediators of c-Myc in this process are the proapoptotic members of the Bcl-2 family. We show here that fibroblasts lacking Bak remain susceptible to c-Myc-induced apoptosis whereas bax-deficient fibroblasts are resistant. However, despite this requirement for Bax, c-Myc activation exerts no detectable effects on Bax expression, localization, or conformation. Moreover, susceptibility to c-Myc-induced apoptosis can be restored in bax-deficient cells by ectopic expression of Bax or by microinjection of a peptide comprising a minimal BH3 domain. Microinjection of BH3 peptide also restores sensitivity to c-Myc-induced apoptosis in p53-deficient primary fibroblasts that are otherwise resistant. By contrast, there is no synergy between BH3 peptide and c-Myc in fibroblasts deficient in both Bax and Bak. We conclude that c-Myc triggers a proapoptotic mitochondrial destabilizing activity that cooperates with proapoptotic members of the Bcl-2 family.

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.

Using a series of insulin-like growth factor I (IGF-I) receptor mutants, we have attempted to define domains required for transmitting the antiapoptotic signal from the receptor and to compare these domains with those required for mitogenesis or transformation. In FL5.12 cells transfected with wild-type IGF-I receptors, IGF-I affords protection from interleukin 3 withdrawal but is not mitogenic. An IGF-I receptor lacking a functional ATP binding site provided no protection from apoptosis. However, receptors mutated at tyrosine residue 950 or in the tyrosine cluster (1131, 1135, and 1136) within the kinase domain remained capable of suppressing apoptosis, although such mutations are known to inactivate transforming and mitogenic functions. In the C terminus of the IGF-I receptor, two mutations, one at tyrosine 1251 and one which replaced residues histidine 1293 and lysine 1294, abolished the antiapoptotic function, whereas mutation of the four serines at 1280 to 1283 did not. Interestingly, receptors truncated at the C terminus had enhanced antiapoptotic function. In Rat-1/ c-MycER fibroblasts, the Y950F mutant and the tyrosine cluster mutant could still provide protection from c-Myc-induced apoptosis, whereas mutant Y1250/1251F could not. These studies demonstrate that the domains of the IGF-I receptor required for its antiapoptotic function are distinct from those required for its proliferation or transformation functions and suggest that domains of the receptor required for inhibition of apoptosis are necessary but not sufficient for transformation.

Amino acids 106 to 143 and 354 to 433 of the human c-myc protein (439 amino acids) were shown to be required for the protein to suppress c-myc gene transcription and were found to exactly overlap with those necessary for c-myc to cooperate with ras oncogenes in the transformation of rat embryo fibroblasts. The essential carboxyl-terminal region harbors structural motifs (a basic region, a helix-loop-helix motif, and a "leucine zipper"), which, in other proteins, can mediate dimerization and sequence-specific DNA binding.

We isolated a series of monoclonal antibodies which were raised against a bacterially expressed protein, bp37v-myb, and coded for by part of the avian v-myb gene. These monoclonal antibodies recognized a range of antigenic specificities on bp37v-myb, and this was reflected in their differing specificities for the gene products of the v-myb, c-myb, and E26 viral oncogenes. One monoclonal antibody recognized, in addition to the v-myb and c-myb gene products, a conserved nuclear protein found in all tested cells. We describe the characterization of these monoclonal antibodies.

Cells of a clone of avian myeloblastosis virus-transformed myeloblasts were induced to differentiate to adherent myelomonocytic cells by treatment with lipopolysaccharide. These adherent cells were subcultured and maintained as a line for more than 6 months with lipopolysaccharide present. Cells of this line were induced to differentiate to nondividing macrophage-like cells by the addition of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. In this way, the following homogeneous cell populations representing three distinct stages of myeloid differentiation were obtained: I, actively dividing myeloblasts that grew in suspension: II, actively dividing adherent cells; and III, fully differentiated nondividing cells resembling macrophages. When the expression of v-myb (the oncogene of avian myeloblastosis virus) was examined in cells of these three differentiation stages, it was found that the protein encoded by v-myb (p45v-myb) continued to be synthesized in similar quantities and showed no obvious alteration (assessed by partial proteolytic digestion and two-dimensional gel electrophoresis) during differentiation. These results show that cells transformed by v-myb can be induced to differentiate without affecting the expression of v-myb and imply that, during differentiation, the effect of v-myb is suppressed by a mechanism other than altered expression of the oncogene.

The retroviral oncogene v-erb-B encodes a truncated version of the receptor for epidermal growth factor. To define the disposition of the v-erb-B protein within cells and across the plasma membrane, we raised antibodies against defined epitopes in the protein and used these in immunofluorescence to analyze cells transformed by v-erb-B. A small fraction of the v-erb-B protein was found on the plasma membrane in a clustered configuration. The bulk of the protein was located in the endoplasmic reticulum and Golgi apparatus. Epitopes near the amino terminus of the v-erb-B protein were displayed on the surface of the cell, whereas epitopes in the protein kinase domain were located exclusively within cells. We conclude that the v-erb-B protein spans the plasma membrane in a manner similar or identical to that of the epidermal growth factor receptor, even though the viral transforming protein does not possess the signal peptide that is thought to direct insertion of the receptor into the membrane.

Six monoclonal antibodies have been isolated from mice immunized with synthetic peptide immunogens whose sequences are derived from that of the human c-myc gene product. Five of these antibodies precipitate p62c-myc from human cells, and three of these five also recognize the mouse c-myc gene product. None of the antibodies sees the chicken p110gag-myc protein. All six antibodies recognize immunoblotted p62c-myc. These reagents also provide the basis for an immunoblotting assay by which to quantitate p62c-myc in cells.