Impaired DNA damage checkpoint response in MIF-deficient mice

Recent studies demonstrated that proinflammatory migration inhibitory factor(MIF) blocks p53-dependent apoptosis and interferes with the tumor suppressor activity of p53. To explore the mechanism underlying this MIF-p53 relationship, we studied spontaneous tumorigenesis in genetically matched p53-/- and MIF-/-p53-/- mice. We show that the loss of MIF expression aggravates the tumor-prone phenotype of p53-/- mice and predisposes them to a broader tumor spectrum, including B-cell lymphomas and carcinomas. Impaired DNA damage response is at the root of tumor predisposition of MIF-/-p53-/- mice. We provide evidence that MIF plays a role in regulating the activity of Cul1-containing SCF ubiquitin ligases. The loss of MIF expression uncouples Chk1/Chk2-responsive DNA damage checkpoints from SCF-dependent degradation of key cell-cycle regulators such as Cdc25A, E2F1 and DP1, creating conditions for the genetic instability of cells. These MIF effects depend on its association with the Jab1/CSN5 subunit of the COP9/CSN signalosome. Given that CSN plays a central role in the assembly of SCF complexes in vivo, regulation of Jab1/CSN5 by MIF is required to sustain optimal composition and function of the SCF complex.     

Multiple functions of Jab1 are required for early embryonic development and growth potential in mice

Jab1 interacts with a variety of signaling molecules and regulates their stability in mammalian cells. As the fifth component of the COP9 signalosome (CSN) complex, Jab1 (CSN5) plays a central role in the deneddylation of the cullin subunit of the Skp1-Cullin-F box protein ubiquitin ligase complex. In addition, a CSN-independent function of Jab1 is suggested but is less well characterized. To elucidate the function of Jab1, we targeted the Jab1 locus by homologous recombination in mouse embryonic stem cells. Jab1-null embryos died soon after implantation. Jab1-/- embryonic cells, which lacked other CSN components, expressed higher levels of p27, p53, and cyclin E, resulting in impaired proliferation and accelerated apoptosis. Jab1 heterozygous mice were healthy and fertile but smaller than their wild-type littermates. Jab1+/- mouse embryonic fibroblast cells, in which the amount of Jab1-containing small subcomplex, but not that of CSN, was selectively reduced, proliferated poorly, showed an inefficient down-regulation of p27 during G1, and was delayed in the progression from G0 to S phase by 3 h compared with the wild-type cells. Most interestingly, in Jab1+/- mouse embryonic fibroblasts, the levels of cyclin E and deneddylated Cul1 were unchanged, and p53 was not induced. Thus, Jab1 controls cell cycle progression and cell survival by regulating multiple cell cycle signaling pathways.     

Identification of Genomic Regions Required for DNA Replication during Drosophila Embryogenesis

A collection of Drosophila deficiency stocks was examined by bromodeoxyuridine (BrdU) labeling of embryos to analyze the DNA replication patterns in late embryogenesis. This permitted us to screen 34% of the genome for genes that when absent in homozygous deficiencies affect the cell cycle or DNA replication. We found three genomic intervals that when deleted result in cessation of DNA replication in the embryo, 39D2-3;E2-F1, 51E and 75C5-7;F1. Embryos deleted for the 75C5-7;F1 region stop DNA replication at the time in embryogenesis when a G(1) phase is added to the mitotic cell cycle and the larval tissues begin to become polytene. Thus, this interval may contain a gene controlling these cell cycle transitions. DNA replication arrests earlier in embryos homozygous for deletions for the other two regions. Analysis of the effects of deletions in the 39D2-3;E2-F1 region on DNA replication showed that the block to DNA replication correlates with deletion of the histone genes. We were able to identify a single, lethal complementation group in 51E, l(2)51Ec, that is responsible for the cessation of replication observed in this interval. Deficiencies that removed one of the Drosophila cdc2 genes and the cyclin A gene had no effect on replication during embryogenesis. Additionally, our analysis identified a gene, pimples, that is required for the proper completion of mitosis in the post-blastoderm divisions of the embryo.     

The cytoplasmic shuttling and subsequent degradation of p27Kip1 mediated by Jab1/CSN5 and the COP9 signalosome complex.

The fifth component of the COP9 signalosome complex, Jab1/CSN5, directly binds to and induces specific down-regulation of the cyclin-dependent kinase inhibitor p27 (p27(Kip1)). Nuclear-cytoplasmic translocation plays an important role because leptomycin B (LMB), a chemical inhibitor of CRM1-dependent nuclear export, prevents p27 degradation mediated by Jab1/CSN5. Here we show that Jab1/CSN5 functions as an adaptor between p27 and CRM1 to induce nuclear export and subsequent degradation. Jab1/CSN5, but not p27, contains a typical leucine-rich nuclear export signal (NES) sequence conserved among different species, through which CRM1 bound to Jab1/CSN5 in an LMB-sensitive manner. Alteration of conserved leucine residues to alanine within Jab1/CSN5-NES abolished the interaction with CRM1 in vitro and impaired LMB-sensitive nuclear export and the ability to induce p27 breakdown in cultured cells. A Jab1/CSN5 truncation mutant lacking NES reversed p27 down-regulation induced by the full-length Jab1/CSN5, indicating that this mutant functions as a dominant negative (DN-Jab1). Introduction of DN-Jab1 into proliferating fibroblasts increased the level of p27 protein, thereby inducing growth arrest of the cells. Random mutagenesis analysis revealed that specific aspartic acid, leucine, and asparagine residues contained in the Jab1/CSN5-binding domain of p27 were required for interaction with Jab1/CSN5 and for down-regulation of p27. Glycerol gradient and cell fractionation experiments showed that at least two different forms of Jab1/CSN5-containing complexes existed within the cell. One is the conventional 450-kDa COP9 signalosome (CSN) complex located in the nucleus, and the other is much smaller (around 100-kDa), containing only a subset of CSN components (CSN4-8 but not CSN1-3), and mainly located in the cytoplasm. Treatment of cells with LMB greatly reduced the level of the smaller complex, suggesting that it originated from the CSN complex by nuclear export. Besides Jab1/CSN5, CSN3, -6, -7, and -8 were capable of inducing p27 down-regulation, when ectopically expressed. These results indicate that cytoplasmic shuttling regulated by Jab1/CSN5 and other CSN components may be a new pathway to control the intracellular abundance of the key cell cycle regulator.     

CSN5/Jab1 controls multiple events in the mammalian cell cycle

The COP9 signalosome (CSN) complex is critical for mammalian cell proliferation and survival, but it is not known how the CSN affects the cell cycle. In this study, MEFs lacking CSN5/Jab1 were generated using a CRE-flox system. MEFs ceased to proliferate upon elimination of CSN5/Jab1. Rescue experiments indicated that the JAMM domain of CSN5/Jab1 was essential. CSN5/Jab1-elimination enhanced the neddylation of cullins 1 and 4 and altered the expression of many factors including cyclin E and p53. CSN5/Jab1-elimination inhibited progression of the cell cycle at multiple points, seemed to initiate p53-independent senescence and increased the ploidy of cells. Thus, CSN5/Jab1 controls different events of the cell cycle, preventing senescence and endocycle as well as the proper progression of the somatic cell cycle.

Structured summary

MINT-8046253: Csn1 (uniprotkb:Q99LD4) physically interacts (MI:0914) with Csn5 (uniprotkb:O35864), Csn8 (uniprotkb:Q8VBV7), Csn3 (uniprotkb:O88543), Csn7b (uniprotkb:Q8BV13) and Csn6 (uniprotkb:O88545) by anti bait coimmunoprecipitation (MI:0006)     

Anti-cancer effects of naturally occurring compounds through modulation of signal transduction and miRNA expression in human colon cancer cells

Much evidence indicates that various naturally occurring compounds have an anti-cancer effect, but the detailed mechanisms are not well understood. In this study, we selected anti-cancer phytochemicals such as epigallocatechin-3-gallate (EGCG), resveratrol (RES) and α-mangostin (α-M), all of which are well-characterized chemopreventive agents. We sought to elucidate the mechanism of their anti-cancer effects and the synergistic effects obtained by combined treatment with the anti-cancer drug 5-fluorouracil (5-FU) in three human colon cancer cell lines. The numbers of viable cells were consistently decreased by the treatment with EGCG, RES or α-M at more than 10 μM in all three cell lines tested. All compounds mainly induced apoptosis and suppressed the PI3K/Akt signaling pathway. Additionally, α-M, which had the greatest PI3K/Akt-suppressing activity, also suppressed MAP kinase (MAPK)/Erk1/2 signaling. Importantly, the combination treatment with RES and 5-FU induced a remarkably synergistic enhancement of growth inhibition and apoptosis through the additional suppression of the MAPK/Erk1/2 signaling pathway in colon cancer DLD-1 cells. Interestingly, RES increased the intracellular expression level of miR-34a, which down-regulated the target gene E2F3 and its downstream Sirt1, resulting in growth inhibition. These findings indicate that these compounds functioned as chemosensitizers when combined with anti-cancer drugs through the modulation of apoptotic and growth-related signaling pathways. Also, RES exerted its anti-cancer activity in part through a newly defined mechanism, i.e., the miR-34a/E2F3/Sirt1 cascade.     

14-3-3 proteins integrate E2F activity with the DNA damage response

The E2F family is composed of at least eight E2F and two DP subunits, which in cells exist as E2F/DP heterodimers that bind to and regulate E2F target genes. While DP-1 is an essential and widespread component of E2F, much less is known about the DP-3 subunit, which exists as a number of distinct protein isoforms that differ in several respects including the presence of a nuclear localisation signal (NLS). We show here that the NLS region of DP-3 harbours a binding site for 14-3-3epsilon, and that binding of 14-3-3epsilon alters the cell cycle and apoptotic properties of E2F. DP-3 responds to DNA damage, and the interaction between DP-3 and 14-3-3epsilon is under DNA damage-responsive control. Further, 14-3-3epsilon is present in the promoter region of certain E2F target genes, and reducing 14-3-3epsilon levels induces apoptosis. These results identify a new level of control on E2F activity and, at a more general level, suggest that 14-3-3 proteins integrate E2F activity with the DNA damage response.     

Inhibition of PI3K by ZSTK474 suppressed tumor growth not via apoptosis but G0/G1 arrest

Phosphoinositide 3-kinase (PI3K) is a potential target in cancer therapy. Inhibition of PI3K is believed to induce apoptosis. We recently developed a novel PI3K inhibitor ZSTK474 with antitumor efficacy. In this study, we have examined the underlying mode of action by which ZSTK474 exerts its antitumor efficacy. In vivo, ZSTK474 effectively inhibited the growth of human cancer xenografts. In parallel, ZSTK474 treatment suppressed the expression of phospho-Akt, suggesting effective PI3K inhibition, and also suppressed the expression of nuclear cyclin D1 and Ki67, both of which are hallmarks of proliferation. However, ZSTK474 treatment did not increase TUNEL-positive apoptotic cells. In vitro, ZSTK474 induced marked G₀/G₁ arrest, but did not increase the subdiploid cells or activate caspase, both of which are hallmarks of apoptosis. These results clearly indicated that inhibition of PI3K by ZSTK474 did not induce apoptosis but rather induced strong G₀/G₁ arrest, which might cause its efficacy in tumor cells.     

E2F1 mediates DNA damage and apoptosis through HCF‐1 and the MLL family of histone methyltransferases

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti‐oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1‐to‐S phase transition, E2F1 associates through a short DHQY sequence with the cell‐cycle regulator HCF‐1 together with the mixed‐lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF‐1‐binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF‐1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF‐1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF‐1‐binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF‐1 association with E2F1 is a regulator of E2F1‐induced apoptosis.