Differential regulation of RNF8-mediated Lys48- and Lys63-based poly-ubiquitylation

Pairing of a given E3 ubiquitin ligase with different E2s allows synthesis of ubiquitin conjugates of different topologies. While this phenomenon contributes to functional diversity, it remains largely unknown how a single E3 ubiquitin ligase recognizes multiple E2s, and whether identical structural requirements determine their respective interactions. The E3 ubiquitin ligase RNF8 that plays a critically important role in transducing DNA damage signals, interacts with E2s UBCH8 and UBC13, and catalyzes both K48- and K63-linked ubiquitin chains. Interestingly, we report here that a single-point mutation (I405A) on the RNF8 polypeptide uncouples its ability in catalyzing K48- and K63-linked ubiquitin chain formation. Accordingly, while RNF8 interacted with E2s UBCH8 and UBC13, its I405A mutation selectively disrupted its functional interaction with UBCH8, and impaired K48-based poly-ubiquitylation reactions. In contrast, RNF8 I405A preserved its interaction with UBC13, synthesized K63-linked ubiquitin chains, and assembled BRCA1 and 53BP1 at sites of DNA breaks. Together, our data suggest that RNF8 regulates K48- and K63-linked poly-ubiquitylation via differential RING-dependent interactions with its E2s UBCH8 and UBC13, respectively.published_or_final_versio

Impact of G 2 checkpoint defect on centromeric instability

Centromeric instability is characterized by dynamic formation of centromeric breaks, deletions, isochromosomes and translocations, which are commonly observed in cancer. So far, however, the mechanisms of centromeric instability in cancer cells are still poorly understood. In this study, we tested the hypothesis that G 2 checkpoint defect promotes centromeric instability. Our observations from multiple approaches consistently support this hypothesis. We found that overexpression of cyclin B1, one of the pivotal genes driving G 2 to M phase transition, impaired G 2 checkpoint and promoted the formation of centromeric aberrations in telomerase-immortalized cell lines. Conversely, centromeric instability in cancer cells was ameliorated through reinforcement of G 2 checkpoint by cyclin B1 knockdown. Remarkably, treatment with KU55933 for only 2.5 h, which abrogated G 2 checkpoint, was sufficient to produce centromeric aberrations. Moreover, centromeric aberrations constituted the major form of structural abnormalities in G 2 checkpoint-defective ataxia telangiectasia cells. Statistical analysis showed that the frequencies of centromeric aberrations in G 2 checkpoint-defective cells were always significantly overrepresented compared with random assumption. As there are multiple pathways leading to G 2 checkpoint defect, our finding offers a broad explanation for the common occurrence of centromeric aberrations in cancer cells. © 2011 Macmillan Publishers Limited All rights reserved.postprin

STK295900, a Dual Inhibitor of Topoisomerase 1 and 2, Induces G<inf>2</inf> Arrest in the Absence of DNA Damage

STK295900, a small synthetic molecule belonging to a class of symmetric bibenzimidazoles, exhibits antiproliferative activity against various human cancer cell lines from different origins. Examining the effect of STK295900 in HeLa cells indicates that it induces G2 phase arrest without invoking DNA damage. Further analysis shows that STK295900 inhibits DNA relaxation that is mediated by topoisomerase 1 (Top 1) and topoisomerase 2 (Top 2) in vitro. In addition, STK295900 also exhibits protective effect against DNA damage induced by camptothecin. However, STK295900 does not affect etoposide-induced DNA damage. Moreover, STK295900 preferentially exerts cytotoxic effect on cancer cell lines while camptothecin, etoposide, and Hoechst 33342 affected both cancer and normal cells. Therefore, STK295900 has a potential to be developed as an anticancer chemotherapeutic agent. © 2013 Kim et al

Tracking the cell cycle origins for escape from topotecan action by breast cancer cells

The anticancer agent topotecan is considered to be S-phase specific. This implies that cancer cells that are not actively replicating DNA could resist the effects of the drug. The cycle specificity of topotecan action was investigated in MCF-7 cells, using time-lapse microscopy to link the initial cell cycle position during acute exposures to topotecan with the antiproliferative consequences for individual cells. The bioactive dose range (0.5–10 μM) for 1-h topotecan exposures was defined by rapid drug delivery and topoisomerase I trapping. Topotecan caused pan-cycle induction and activation of p53. Lineage analysis of the time-lapse sequences identified cells initially in S-phase and G2, and defined the time to mitosis for cells originating from G2, S-phase and G1. Topotecan prevented all mitoses from S-phase cells and G1 cells (half-maximal effects at 0.14 μM and 0.96 μM, respectively). No dose of topotecan completely prevented mitosis among G2 cells, and at saturating doses of topotecan about half the cells of G2 origin continued dividing (the half-maximal effects was at 0.31 μM). Overall, topotecan differentially targeted G1-, S- and G2-phase cells, but many G2 cells were resistant to topotecan, presenting a clear route for cell cycle-mediated drug resistance

Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer

Background: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis

Dual mechanism of daunorubicin-induced cell death in both sensitive and MDR-resistant HL-60 cells

Exposure of some acute myeloid leukaemia (AML) cells to daunorubicin leads to rapid cell death, whereas other AML cells show natural drug resistance. This has been attributed to expression of functional P-glycoprotein resulting in reduced drug accumulation. However, it has also been proposed that P-glycoprotein-expressing multidrug-resistant (MDR) cells are inherently defective for apoptosis. To distinguish between these different possibilities, we have compared the cell death process in a human AML cell line (HL-60) with a MDR subline (HL-60/Vinc) at doses that yield either similar intracellular daunorubicin concentrations or comparable cytotoxicity. Adjustment of the dose to obtain the same intracellular drug accumulation in the two cell lines did not result in equal cytotoxicity, suggesting the presence of additional resistance mechanisms in the P-glycoprotein-expressing HL-60/Vinc cells. However, at equitoxic doses, similar cell death pathways were observed. In HL-60 cells, daunorubicin induced rapid apoptosis at 0.5–1 μM and delayed mitotic cell death at 0.1 μM. These concentrations are within the clinical dose range. Similarly, HL-60/Vinc cells underwent apoptosis at 50–100 μM daunorubicin and mitotic cell death at 10 μM. These results show, for the first time, that anthracyclines can induce cell death by a dual mechanism in both sensitive and MDR cells. Our results also show that not only the cytotoxicity, but also the kinetics and mechanism of cell death, are dose dependent. Interestingly, regrowth was observed only in association with delayed cell death and the formation of enlarged, often polyploid, cells with micronucleation, suggesting that morphological criteria may be useful to evaluate treatment efficacy in patients with myeloid leukaemias. © 1999 Cancer Research Campaig

Theoretical Analysis of the Stress Induced B-Z Transition in Superhelical DNA

We present a method to calculate the propensities of regions within a DNA molecule to transition from B-form to Z-form under negative superhelical stresses. We use statistical mechanics to analyze the competition that occurs among all susceptible Z-forming regions at thermodynamic equilibrium in a superhelically stressed DNA of specified sequence. This method, which we call SIBZ, is similar to the SIDD algorithm that was previously developed to analyze superhelical duplex destabilization. A state of the system is determined by assigning to each base pair either the B- or the Z-conformation, accounting for the dinucleotide repeat unit of Z-DNA. The free energy of a state is comprised of the nucleation energy, the sequence-dependent B-Z transition energy, and the energy associated with the residual superhelicity remaining after the change of twist due to transition. Using this information, SIBZ calculates the equilibrium B-Z transition probability of each base pair in the sequence. This can be done at any physiologically reasonable level of negative superhelicity. We use SIBZ to analyze a variety of representative genomic DNA sequences. We show that the dominant Z-DNA forming regions in a sequence can compete in highly complex ways as the superhelicity level changes. Despite having no tunable parameters, the predictions of SIBZ agree precisely with experimental results, both for the onset of transition in plasmids containing introduced Z-forming sequences and for the locations of Z-forming regions in genomic sequences. We calculate the transition profiles of 5 kb regions taken from each of 12,841 mouse genes and centered on the transcription start site (TSS). We find a substantial increase in the frequency of Z-forming regions immediately upstream from the TSS. The approach developed here has the potential to illuminate the occurrence of Z-form regions in vivo, and the possible roles this transition may play in biological processes

RNA-Binding Protein Musashi1 Modulates Glioma Cell Growth through the Post-Transcriptional Regulation of Notch and PI3 Kinase/Akt Signaling Pathways

Musashi1 (MSI1) is an RNA-binding protein that plays critical roles in nervous-system development and stem-cell self-renewal. Here, we examined its role in the progression of glioma. Short hairpin RNA (shRNA)-based MSI1-knock down (KD) in glioblastoma and medulloblastoma cells resulted in a significantly lower number of self renewing colony on day 30 (a 65% reduction), compared with non-silencing shRNA-treated control cells, indicative of an inhibitory effect of MSI1-KD on tumor cell growth and survival. Immunocytochemical staining of the MSI1-KD glioblastoma cells indicated that they ectopically expressed metaphase markers. In addition, a 2.2-fold increase in the number of MSI1-KD cells in the G2/M phase was observed. Thus, MSI1-KD caused the prolongation of mitosis and reduced the cell survival, although the expression of activated Caspase-3 was unaltered. We further showed that MSI1-KD glioblastoma cells xenografted into the brains of NOD/SCID mice formed tumors that were 96.6% smaller, as measured by a bioluminescence imaging system (BLI), than non-KD cells, and the host survival was longer (49.3±6.1 days vs. 33.6±3.6 days; P<0.01). These findings and other cell biological analyses suggested that the reduction of MSI1 in glioma cells prolonged the cell cycle by inducing the accumulation of Cyclin B1. Furthermore, MSI1-KD reduced the activities of the Notch and PI3 kinase-Akt signaling pathways, through the up-regulation of Numb and PTEN, respectively. Exposure of glioma cells to chemical inhibitors of these pathways reduced the number of spheres and living cells, as did MSI1-KD. These results suggest that MSI1 increases the growth and/or survival of certain types of glioma cells by promoting the activation of both Notch and PI3 kinase/Akt signaling

Reversal of MDR1-associated resistance to topotecan by PAK-200S, a new dihydropyridine analogue, in human cancer cell lines

Recent data suggest that expression of the membrane P170-glycoprotein (P-gp) may confer resistance to the topoisomerase- I-interactive agent topotecan. The present study describes the cellular effects of a new dihydropyridine analogue, PAK-200S, on P-gp-mediated resistance to topotecan in human breast and ovarian tumour cells. PAK-200S at a non-cytotoxic concentration of 2.0 μM completely reversed resistance to topotecan in P-gp-expressing MCF-7/adr (breast) and A2780/Dx5 (ovarian) tumour cells, respectively, with no effects on parental cells. Cellular pharmacokinetic studies by reversed-phase high-performance liquid chromatography analysis showed significantly lower cellular drug concentrations of the pharmacologically active closed-ring lactone of topotecan in multidrug-resistant cells than in parental cells. PAK-200S was effective in restoring the cellular lactone concentrations of topotecan in resistant MCF-7/adr cells to levels comparable to those obtained in parental cells. Furthermore, exposure of MCF-7/adr cells to topotecan in the presence of PAK-200S significantly increased the induction of protein-linked DNA breaks. PAK-200S did not alter nuclear topoisomerase I-mediated ex vivo pBR322 DNA plasmid unwinding activity and topoisomerase-I protein expression. These results suggest that reversal of P-gp-mediated resistance to topotecan by PAK-200S was related to the restoration of cellular drug concentrations of the active lactone form of topotecan rather than a direct effect on topoisomerase-I function. © 1999 Cancer Research Campaig

Pan-cancer analysis of whole genomes

Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale(1-3). Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4-5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter(4); identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation(5,6); analyses timings and patterns of tumour evolution(7); describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity(8,9); and evaluates a range of more-specialized features of cancer genomes(8,10-18).Peer reviewe