Aside from the well-established roles of c-Myc in the regulation of cell cycle, differentiation, and apoptosis, a recent picture is beginning to emerge linking c-Myc to the regulation of metabolic pathways. Here, we define a further function for c-Myc in determining cellular redox balance, identifying glutathione (GSH) as the leading molecule mediating this process. The link between c-Myc and GSH is gamma-glutamyl-cysteine synthetase (gamma-GCS), the rate-limiting enzyme catalyzing GSH biosynthesis. Indeed, c-Myc transcriptionally regulates gamma-GCS by binding and activating the promoters of both gamma-GCS heavy and light subunits. Exposure to H2O2 enhances c-Myc recruitment to gamma-GCS regulatory regions through ERK-dependent phosphorylation. Phosphorylation at Ser-62 is required for c-Myc recruitment to gamma-GCS promoters and determines the cellular response to oxidative stress induced by different stimuli. Thus, the c-Myc phosphorylation-dependent activation of the GSH-directed survival pathway can contribute to oxidative stress resistance in tumor cells, which generally exhibit deregulated c-Myc expression.

Deubiquitinating enzymes can prevent the destruction of protein substrates prior to proteasomal degradation. The ubiquitin-specific protease 2a (USP2a) deubiquitinates the antiapoptotic proteins Fatty Acid Synthase and Mdm2. Here, we show that when USP2a is overexpressed in nontransformed cells, it exhibits oncogenic behavior both in vitro and in vivo and prevents apoptosis induced by chemotherapeutic agents. Notably, USP2a silencing in several human cancer cell lines results in apoptosis. Gene set enrichment analysis, which focuses on groups of genes sharing biological function or regulatory pathways, was done on microarray expression data from human prostate cancers. The cell death-related gene set, as well as a selected cluster of validated p53 target genes, were significantly enriched in the low USP2a expression group of tumors. Conversely, genes implicated in fatty acid metabolism were significantly associated with tumors expressing high USP2a (44%). The expression profile analysis is consistent with the effects of USP2a on its known targets, i.e., Fatty Acid Synthase and Mdm2, defining a subset of prostate tumors resistant to apoptosis. USP2a thus represents a therapeutic target in prostate cancer. (Cancer Res 2006; 66(17): 8625-32)

DNA tumor virus oncoproteins bind and inactivate Rb by interfering with the Rb/HDAC1 interaction. Che-1 is a recently identified human Rb binding protein that inhibits the Rb growth suppressing function. Here we show that Che-1 contacts the Rb pocket region and competes with HDAC1 for Rb binding site, removing HDAC1 from the Rb/E2F complex in vitro and from the E2F target promoters in vivo. Che-1 overexpression activates DNA synthesis in quiescent NIH-3T3 cells through HDAC1 displacement. Consistently, Che-1-specific RNA interference affects E2F activity and cell proliferation in human fibroblasts but not in the pocket protein-defective 293 cells. These findings indicate the existence of a pathway of Rb regulation supporting Che-1 as the cellular counterpart of DNA tumor virus oncoproteins.

This study had two goals: 1) to evaluate the biological effect of the novel pentacyclic acridine 3, 11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) on human melanoma lines possessing long telomeres, and 2) to elucidate the relationship between G-quadruplex-based telomerase inhibitor-induced cellular effects and telomere length/dysfunction. The cellular pharmacological effects of RHPS4 have been evaluated by treating melanoma lines with increasing concentrations of RHPS4. A dose-dependent inhibition of cell proliferation was observed in all the lines during short-term treatment. Flow cytometric analysis demonstrated that RHPS4 induced a dose-dependent accumulation of cells in the S-G 2 /M phase of cell cycle. The RHPS4-induced cell cycle alteration was irreversible even at low doses, and the cells died from apoptosis. At high RHPS4 concentration, apoptosis was accompanied by the induction of a senescence phenotype: large cell size, vacuolated cytoplasm, and ␤-galactosidase activity. The shortterm biological activity of RHPS4 was not caused by telomere shortening, but it was associated with telomere dysfunction, in terms of presence of telomeric fusions, polynucleated cells, and typical images of telophase bridge. In conclusion, our results demonstrate that the G-quadruplex ligand RHPS4 can function in a telomere length-independent manner through its ability to cause telomere-capping alteration.