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A report on the biannual Swiss Institute for Experimental Cancer Research (ISREC) Symposium on the Cell and Molecular Biology of Cancer, Lausanne, Switzerland, 19-22 January 2005.

Activation of the protein kinase p34cdc2 is required for entry into meiotic or mitotic M phase in all eukaryotic cells. One important mechanism regulating the activity of p34cdc2 during the cell cycle is based on phosphorylation/dephosphorylation. Avian p34cdc2 is phosphorylated on threonine 14 (Thr14), tyrosine 15 (Tyr15), threonine 161 (Thr161) and serine 277 (Ser277). Dephosphorylation of both Thr14 and Tyr15 is required for activation of p34cdc2 at the G2/M transition, indicating that phosphorylation of these residues negatively regulates p34cdc2 activity. Conversely, phosphorylation of Thr161 is required for kinase activity. Whether modification of this residue is due to intramolecular autophosphorylation or to the action of an as yet unidentified kinase remains unresolved. Likewise, the role of phosphorylation of p34cdc2 on Ser277 during G1 phase of the cell cycle remains to be determined. The function of p34cdc2 is regulated also by cell cycle-dependent complex formation with cyclin proteins. We found that chicken cyclin B2 undergoes a striking redistribution from the cytoplasm to the nucleus just prior to the onset of mitosis. Expression of a non-destructible cyclin B2 mutant causes HeLa cells to arrest in mitosis. Frequently, arrested cells displayed multiple mitotic spindles.

Myc proteins are powerful proto-oncoproteins and important promoters of growth and proliferation during normal development. They are thought to exercise their effects upon binding to their partner protein Max, and their activities are largely antagonized by complexes of Max with Mnt or an Mxd family protein. Although the biological functions of Myc, Mxd and Mnt have been intensively studied, comparatively little is known about the in vivo role of Max. Here we generate Max loss-of-function and reduction-of-function mutations in Drosophila melanogaster to address the contribution of Max to Myc-dependent growth control. We find that many biological activities of Myc do not, or only partly, require the association with Max--for example, the control of endoreplication and cell competition-and that a Myc mutant that does not interact with Max retains substantial biological activity. We further show that Myc can control RNA polymerase III independently of Max, which explains some of Myc's observed biological activities. These studies show the ability of Myc to function independently of Max in vivo and thus change the current model of Max network function.

Myc proto-oncoproteins are important regulators of growth and proliferation in development. Their functions have been evolutionarily conserved from insects to vertebrates, although the sequence conservation is limited to a few short domains. Here, we analyze the requirement for the most highly conserved domains, called Myc boxes 2 and 3 (MB2 and MB3), and for the weakly conserved N-terminus for the biological activity of the single Drosophila Myc protein in the animal in vivo. We find that a Myc mutant lacking the N-terminus retains very little activity, whereas Myc transgenes carrying a deletion of MB3 have a moderately increased ability to promote growth and apoptosis; mutation of MB2 reduces transcriptional output and the biological activities of Myc. Surprisingly though, Myc without MB2 retains enough activity to partially rescue the lethality of a Myc null mutation. Thus, although MB2 and MB3 are highly conserved in evolution, loss of either domain has comparatively mild consequences on Myc activity in vivo.

The proto-oncogene Myc is already known to affect many cellular processes, but recent experiments in the fruit fly Drosophila melanogaster have revealed yet a new facet of Myc. Neighboring cells were shown to compare their Myc levels and the losers (cells with lower Myc activity) were actively eliminated. This phenomenon is called ‘‘cell competition,’’ and it seems to be part of a developmental size and quality control program. Subversion of this mechanism may contribute to the transforming powers of Myc and possibly other oncogenes. (Cancer Res 2005; 65(15): 6485-7) Background In recent years, the modest fruit fly Drosophila melanogaster has become a popular model system for the analysis of cellular growth control and organ size determination during animal development. For their studies, many biologists have focused on the so-called wing imaginal discs, the precursor organs of adult wings and thoraxes. Wing imaginal discs originate from a group of 40 to 50 cells that are set aside at the end of embryogenesis. During the 4 days of larval development, these cells multiply 1,000-fold to form the mature imaginal disc, which consists mainly of a columnar epithelial monolayer (1). The cellular growth and cell cycle characteristics during this proliferative phase closely resemble those of vertebrate cells, inasmuch as similar regulatory proteins have been found to function in both situations. In contrast to cells cultured on plastic, however, imaginal disc cells are embedded in an intact tissue and subject to physiologic short- and long-range signals that cannot be observed in vitro. One phenomenon based on such signals was identified 30 years ago: cell competition. Cell competition was observed with a group of mutations called Minutes (M; ref. 2). Minutes are mutations in ribosomal protein genes (3) that are characterized by recessive lethality and by a dominant growth defect. Thus, heterozygous M+/ flies are delayed in their development and take longer to reach their normal size, a reflection of the slower growth rate of M+/ cells. Importantly, however, M+/ cells are viable and can give rise to almost normal-looking animals. In striking contrast, when clones of slow-growing M+/ cells are generated in an animal that otherwise consists of wild-type cells, the M+/ cells are actively eliminated—a process dubbed ‘‘cell competition’’; although such clones can be observed shortly after they have been induced, within 2 days no more surviving M+/ cells can be seen (2). Key Finding A very similar growth defect was recently found to be associated with the sole Drosophila orthologue of the protooncogene Myc, dMyc. Whereas dmyc is an essential gene, hypomorphic dmyc mutations are viable and only characterized by a modest growth defect. However, when clones of cells carrying such a hypomorphic dmyc mutation are surrounded by phenotypically wild-type cells, they suffer from the same type of cell competition as seen for M+/ cells (4). Interestingly, this cell competition is not simply caused by cellular defects associated with the dmyc mutation: Even wild-type cells were shown to be competed when they are surrounded by cells overexpressing dMyc, suggesting that cells somehow compare their dMyc level to that of their neighbors and it is this relative dMyc level that determines whether a cell is competed out of existence (5, 6); importantly, as little as 2-fold differences in dMyc levels are already sufficient to trigger cell competition. This dMyc-dependent cell competition shares two characteristics with the competition of M+/ cells by wild-type cells. First, both only act over a short distance—wildtype cells are only competed up to eight cell diameters from dMyc-overexpressing cells. Second, this cell competition does not function across the boundary between the anterior and the posterior compartment of the wing disc (roughly corresponding to two halves of the wing disc; i.e., wild-type cells in the posterior compartment are not competed by dMyc-overexpressing cells situated in the anterior compartment). The molecular basis for these features is not clear.

Myc is a transcription factor with diverse biological effects ranging from the control of cellular proliferation and growth to the induction of apoptosis. Here we present a comprehensive analysis of the transcriptional targets of the sole Myc ortholog in Drosophila melanogaster, dMyc. We show that the genes that are down-regulated in response to dmyc inhibition are largely identical to those that are up-regulated after dMyc overexpression and that many of them play a role in growth control. The promoter regions of these targets are characterized by the presence of the E-box sequence CACGTG, a known dMyc binding site. Surprisingly, a large subgroup of (functionally related) dMyc targets contains a single E-box located within the first 100 nucleotides after the transcription start site. The relevance of this E-box and its position was confirmed by a mutational analysis of a selected dMyc target and by the observation of its evolutionary conservation in a different Drosophila species, Drosophila pseudoobscura. These observations raise the possibility that a subset of Myc targets share a distinct regulatory mechanism.