In response to DNA damage eukaryotic cells initiate a complex signalling pathway, termed the DNA damage response (DDR), which coordinates cell cycle arrest with DNA repair. Studies have shown that oncogene-induced senescence, which provides a barrier to tumour development, involves activation of the DDR1–3. Using a genome-wide RNA interference (RNAi) screen, we have identified 17 factors required for oncogenic BRAF to induce senescence in primary fibroblasts and melanocytes4. One of these factors is an F-box protein, FBXO31, a candidate tumour suppressor encoded in 16q24.3, a region in which there is loss of heterozygosity in breast, ovarian, hepatocellular and prostate cancers5–9. Here we study the cellular role of FBXO31, identify its target substrate and elucidate the basis for its growth inhibitory activity. We show that ectopic expression of FBXO31 acts through a proteasome-directed pathway to mediate degradation of cyclin D1, an important regulator of G1 to S phase progression, resulting in G1 arrest. Cyclin D1 degradation results from a direct interaction with FBXO31 and is dependent upon the F-box motif of FBXO31 and phosphorylation of cyclin D1 at threonine-286, which is known to be required for cyclin D1 proteolysis. The involvement of the DDR in oncogene-induced senescence prompted us to investigate the role of FBXO31 in DNA repair. We find that DNA damage induced by γ-irradiation results in increased FBXO31 levels, which requires phosphorylation of FBXO31 by the DDR-initiating kinase, ATM. Significantly, RNAi-mediated knockdown of FBXO31 prevents cells from undergoing efficient G1 arrest following γ-irradiation and dramatically increases sensitivity to DNA damage. Finally, we show that diverse DNA damaging agents all result in a large increase in FBXO31 levels, indicating that induction of FBXO31 is a general response to genotoxic stress. Our results reveal FBXO31 as a regulator of the G1/S transition that is specifically required for DNA damage-induced growth arrest.

Bacterial diseases are among the leading causes of human death. The development of antibiotic resistance greatly contributes to the high mortality rate, and thus, the discovery of antibacterial drugs with novel mechanisms of action is needed. In this study, we found that sanguinarine, a benzophenanthridine alkaloid, strongly induced filamentation in both Gram-positive and Gram-negative bacteria and prevented bacterial cell division by inhibiting cytokinesis. Sanguinarine did not perturb the membrane structure in Escherichia coli. However, it perturbed the cytokinetic Z-ring formation in E. coli. In addition, sanguinarine strongly reduced the frequency of the occurrence of Z rings/micrometer of Bacillus subtilis length but did not alter the number of nucleoids/micrometer of cell length. The results suggested that sanguinarine inhibited cytokinesis in B. subtilis by inhibiting Z-ring formation without affecting nucleoid segregation. Sanguinarine inhibited the assembly of purified FtsZ and reduced the bundling of FtsZ protofilaments in vitro. Further, the interaction of sanguinarine to FtsZ was investigated using size-exclusion chromatography, an extrinsic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, and tryptophan fluorescence of mutated FtsZ (Y371W). Sanguinarine was found to bind to FtsZ with a dissociation constant of 18-30 microM. The results together show that sanguinarine inhibits bacterial division by perturbing FtsZ assembly dynamics in the Z ring and provide evidence in support of the hypothesis that the assembly and bundling of FtsZ play a critical role in bacterial cytokinesis. The results suggest that sanguinarine may be used as a lead compound to develop FtsZ-targeted antibacterial agents.

The antifungal drug griseofulvin inhibits mitosis strongly in fungal cells and weakly in mammalian cells by affecting mitotic spindle microtubule (MT) function. Griseofulvin also blocks cell-cycle progression at G2͞M and induces apoptosis in human tumor cell lines. Despite extensive study, the mechanism by which the drug inhibits mitosis in human cells remains unclear. Here, we analyzed the ability of griseofulvin to inhibit cell proliferation and mitosis and to affect MT polymerization and organization in HeLa cells together with its ability to affect MT polymerization and dynamic instability in vitro. Griseofulvin inhibited cell-cycle progression at prometaphase͞anaphase of mitosis in parallel with its ability to inhibit cell proliferation. At its mitotic IC 50 of 20 M, spindles in blocked cells displayed nearly normal quantities of MTs and MT organization similar to spindles blocked by more powerful MTtargeted drugs. Similar to previously published data, we found that very high concentrations of griseofulvin (>100 M) were required to inhibit MT polymerization in vitro. However, much lower drug concentrations (1-20 M) strongly suppressed the dynamic instability behavior of the MTs. We suggest that the primary mechanism by which griseofulvin inhibits mitosis in human cells is by suppressing spindle MT dynamics in a manner qualitatively similar to that of much more powerful antimitotic drugs, including the vinca alkaloids and the taxanes. In view of griseofulvin's lack of significant toxicity in humans, we further suggest that it could be useful as an adjuvant in combination with more powerful drugs for the treatment of cancer.cancer chemotherapy ͉ mitosis

SUMMARY The RASSF1A tumor suppressor gene is epigenetically silenced in a variety of cancers. Here we perform a genome-wide human shRNA screen and find that epigenetic silencing of RASSF1A requires the homeobox protein HOXB3. We show that HOXB3 binds to the DNA methyltransferase DNMT3B gene and increases its expression. DNMT3B, in turn, is recruited to the RASSF1A promoter, resulting in hypermethylation and silencing of RASSF1A expression. DNMT3B recruitment is facilitated through interactions with Polycomb repressor complex 2 and MYC, which is bound to the RASSF1A promoter. Mouse xenograft experiments indicate that the oncogenic activity of HOXB3 is due, at least in part, to epigenetic silencing of RASSF1A. Expression analysis in human lung adenocarcinoma samples reveals that RASSF1A silencing strongly correlates with over-expression of HOXB3 and DNMT3B. Analysis of human cancer cell lines indicates that the RASSF1A epigenetic silencing mechanism described here may be common in diverse cancer types.

The Bcl-2 family of proteins consists of both antiapoptotic and proapoptotic factors, which share sequence homology within conserved regions known as Bcl-2 homology domains.

The tumor suppressor p53 plays a critical role in maintaining genomic stability. In response to genotoxic stress, p53 levels increase and induce cell-cycle arrest, senescence, or apoptosis, thereby preventing replication of damaged DNA. In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that directly interacts with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome. Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Because of the important role of MDM2 in modulating p53 function, it is critical to understand how MDM2 levels are regulated. Here we show that the F-box protein FBXO31, a candidate tumor suppressor encoded in 16q24.3 for which there is loss of heterozygosity in various solid tumors, is responsible for promoting MDM2 degradation. Following genotoxic stress, FBXO31 is phosphorylated by the DNA damage serine/ threonine kinase ATM, resulting in increased levels of FBXO31. FBXO31 then interacts with and directs the degradation of MDM2, which is dependent on phosphorylation of MDM2 by ATM. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage.O ne of the most fundamental and extensively studied anticancer mechanisms is the large increase in the levels of the tumor suppressor p53 that occurs following DNA damage (reviewed in ref. 1). The increased p53 then mediates growth arrest and/or apoptosis. The importance of this anticancer mechanism is highlighted by the mutation or functional inactivation of the p53 gene in more than 50% of human cancers (2, 3).In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that interacts directly with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome (reviewed in ref. 4). Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Originally it was proposed that MDM2 degradation was caused by auto-ubiquitination; however, subsequent experiments showed that the E3 ubiquitin ligase activity of MDM2 is not required for its degradation (5).We originally identified the F-box protein FBXO31 in an RNAi screen as one of 17 factors required for oncogenic BRAF to induce senescence in primary human cells (6). F-box proteins are best known for their role as the substrate-recognition components of the SKP1/CUL1/F-box protein (SCF) class of E3 ubiquitin ligases (7). The F-box motif is responsible for the ability of F-box proteins to interact with the SCF complex and to promote ubiquitination of their targets (8).One of the other genes we isolated in our original RNAi screen was p53 (6), raising the possibility that FBXO31 and p53 function in a...

The changes in the far-UV CD signal, intrinsic tryptophan fluorescence and bilirubin absorbance showed that the guanidine hydrochloride (GdnHCl)-induced unfolding of a multidomain protein, human serum albumin (HSA), followed a two-state process. However, using environment sensitive Nile red fluorescence, the unfolding and folding pathways of HSA were found to follow a three-state process and an intermediate was detected in the range 0.25-1.5 M GdnHCl. The intermediate state displayed 45% higher fluorescence intensity than that of the native state. The increase in the Nile red fluorescence was found to be due to an increase in the quantum yield of the HSA-bound Nile red. Low concentrations of GdnHCl neither altered the binding affinity of Nile red to HSA nor induced the aggregation of HSA. In addition, the secondary structure of HSA was not perturbed during the first unfolding transition ( <1.5 M GdnHCl); however, the secondary structure was completely lost during the second transition. The data together showed that the half maximal loss of the tertiary structure occurred at a lower GdnHCl concentration than the loss of the secondary structure. Further kinetic studies of the refolding process of HSA using multiple spectroscopic techniques showed that the folding occurred in two phases, a burst phase followed by a slow phase. An intermediate with native-like secondary structure but only a partial tertiary structure was found to form in the burst phase of refolding. Then, the intermediate slowly folded into the native state. An analysis of the refolding data suggested that the folding of HSA could be best explained by the framework model.

Cu(II) complexes of three tridentate ligands, L(1), L(2) and L(3), [L(1), N-((1H-imidazole-2-yl)methyl)-2-(pyridine-2-yl)ethanamine; L(2), N-((1-methyl-1H-imidazole-2-yl)methyl)-2-(pyridine-2-yl)ethanamine; L(3), 2-(pyridine-2-yl)-N-((pyridine-2-yl)methyl)ethanamine] respectively, were synthesized and characterized. The single crystal X-ray structure of complex 1 reveals the pseudo octahedral coordination geometry around the copper center. Absorption and fluorescence experimental evidence show good DNA binding propensity (in the order of 10(5) M(-1)) of the complexes. Thermal denaturation and circular dichroism (CD) analyses reveal minor structural changes of calf thymus (CT) DNA in presence of complexes and groove and/or surface binding of the complexes to CT-DNA. Kinetic DNA cleavage assay shows pseudo-first-order kinetic reaction between the complex and supercoiled (SC) DNA. In addition, mechanistic SC DNA cleavage results show higher DNA cleavage activity in presence of reducing agent, due to the presence of hydroxyl radicals. In vitro cytotoxicity assay of the complexes demonstrate that the complexes have low toxicity for different cancer cell lines and IC(50) values were between 37 and 156 μM.