Mutant p53 cooperates with the SWI/SNF chromatin remodeling complex to regulate VEGFR2 in breast cancer cells.

Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.

Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.

Mutations in ANKRD11 have recently been reported to cause KBG syndrome, an autosomal dominant condition characterized by intellectual disability (ID), behavioral problems, and macrodontia. To understand the pathogenic mechanism that relates ANKRD11 mutations with the phenotype of KBG syndrome, we studied the cellular characteristics of wild-type ANKRD11 and the effects of mutations in humans and mice. We show that the abundance of wild-type ANKRD11 is tightly regulated during the cell cycle, and that the ANKRD11 C-terminus is required for the degradation of the protein. Analysis of 11 pathogenic ANKRD11 variants in humans, including six reported in this study, and one reported in the Ankrd11 (Yod/+) mouse, shows that all mutations affect the C-terminal regions and that the mutant proteins accumulate aberrantly. In silico analysis shows the presence of D-box sequences that are signals for proteasome degradation. We suggest that ANKRD11 C-terminus plays an important role in regulating the abundance of the protein, and a disturbance of the protein abundance due to the mutations leads to KBG syndrome.

Transgenic black soldier flies for production of carotenoids.

The black soldier fly (BSF), Hermetia illucens, has gained traction recently as a means to achieve closed-loop production cycles. BSF can subsist off mammalian waste products and their consumption of such waste in turn generates compost that can be used in agricultural operations. Their environmental impact is minimal and BSF larvae are edible, with a nutritional profile high in protein and other essential vitamins. Therefore, it is conceivable to use BSF as a mechanism for both reducing organic waste and maintaining a low-impact food source for animal livestock or humans. The main drawback to BSF as a potential human food source is they are deficient in fat-soluble vitamins such as Vitamins A, D, and E. While loading BSF with essential vitamins may be achieved via diet-based interventions, this undercuts the goal of a closed-loop as specialized diets would require additional supply chains. An alternative is to genetically engineer BSF that can synthesize these essential vitamins. Here we describe a BSF line that has been engineered with the two main carotenoid biosynthetic genes, CarRA and CarB for production of provitamin carotenoids within the Vitamin A family. Our data describe the manipulation of the BSF genome to insert transgenes for expression of functional protein products.

Bioassay-Guided Fractionation of Pittosporum angustifolium and Terminalia ferdinandiana with Liquid Chromatography Mass Spectroscopy and Gas Chromatography Mass Spectroscopy Exploratory Study.

Bioprospecting native Australian plants offers the potential discovery of latent and novel bioactive compounds. The promising cytotoxic and antibacterial activity of methanolic extracts of Pittosporum angustifolium and Terminalia ferdinandiana led to further fractionation and isolation using our laboratory's bioassay-guided fractionation protocol. Hence, the aim of this study was to further evaluate the bioactivity of the fractions and subfractions and characterize bioactive compounds using liquid chromatography mass spectroscopy (LC-MS/MS) and gas chromatography MS (GC-MS). Compounds tentatively identified in P. angustifolium Fraction 1 using LC-ESI-QTOF-MS/MS were chlorogenic acid and/or neochlorogenic acid, bergapten, berberine, 8'-epitanegool and rosmarinic acid. GC-MS analysis data showed the presence of around 100 compounds, mainly comprising carboxylic acids, sugars, sugar alcohols, amino acids and monoalkylglycerols. Furthermore, the fractions obtained from T. ferdinandiana flesh extracts showed no cytotoxicity, except against HT29 cell lines, and only Fraction 2 exhibited some antibacterial activity. The reduced bioactivity observed in the T. ferdinandiana fractions could be attributed to the potential loss of synergy as compounds become separated within the fractions. As a result, the further fractionation and separation of compounds in these samples was not pursued. However, additional dose-dependent studies are warranted to validate the bioactivity of T. ferdinandiana flesh fractions, particularly since this is an understudied species. Moreover, LC-MS/GC-MS studies confirm the presence of bioactive compounds in P. angustifolium Fraction 1/subfractions, which helps to explain the significant acute anticancer activity of this plant. The screening process designed in this study has the potential to pave the way for developing scientifically validated phytochemical/bioactivity information on ethnomedicinal plants, thereby facilitating further bioprospecting efforts and supporting the discovery of novel drugs in modern medicine.

Synthesis and extended activity of triazole-containing macrocyclic protease inhibitors.

Peptide-derived protease inhibitors are an important class of compounds with the potential to treat a wide range of diseases. Herein, we describe the synthesis of a series of triazole-containing macrocyclic protease inhibitors pre-organized into a ß-strand conformation and an evaluation of their activity against a panel of proteases. Acyclic azido-alkyne-based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpain II, cathepsin L and S, and the 20S proteasome chymotrypsin-like activity. Some of the first examples of highly potent macrocyclic inhibitors of cathepsin S were identified. These adopt a well-defined ß-strand geometry as shown by NMR spectroscopy, X-ray analysis, and molecular docking studies.

Development of a novel cell-based assay system EPISSAY for screening epigenetic drugs and liposome formulated decitabine.

BACKGROUND: Despite the potential of improving the delivery of epigenetic drugs, the subsequent assessment of changes in their epigenetic activity is largely dependent on the availability of a suitable and rapid screening bioassay. Here, we describe a cell-based assay system for screening gene reactivation. METHODS: A cell-based assay system (EPISSAY) was designed based on a silenced triple-mutated bacterial nitroreductase TMnfsB fused with Red-Fluorescent Protein (RFP) expressed in the non-malignant human breast cell line MCF10A. EPISSAY was validated using the target gene TXNIP, which has previously been shown to respond to epigenetic drugs. The potency of a epigenetic drug model, decitabine, formulated with PEGylated liposomes was also validated using this assay system. RESULTS: Following treatment with DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors such as decitabine and vorinostat, increases in RFP expression were observed, indicating expression of RFP-TMnfsB. The EPISSAY system was then used to test the potency of decitabine, before and after PEGylated liposomal encapsulation. We observed a 50% higher potency of decitabine when encapsulated in PEGylated liposomes, which is likely to be due to its protection from rapid degradation. CONCLUSIONS: The EPISSAY bioassay system provides a novel and rapid system to compare the efficiencies of existing and newly formulated drugs that reactivate gene expression.

Bioassay Guided Fractionation Protocol for Determining Novel Active Compounds in Selected Australian Flora.

A large variety of unique and distinct flora of Australia have developed exceptional survival methods and phytochemicals and hence may provide a significant avenue for new drug discovery. This study proposes a bioassay guided fractionation protocol that maybe robust and efficient in screening plants with potential bioactive properties and isolating lead novel compounds. Hence, five native Australian plants were selected for this screening process, namely Pittosporum angustifolium (Gumbi gumbi), Terminalia ferdinandiana (Kakadu plum, seeds (KPS), and flesh (KPF)), Cupaniopsis anacardioides (Tuckeroo, seeds (TKS) and flesh (TKF)), Podocarpus elatus (Illawarra plum, seeds (IPS) and flesh (IPF)) and Pleiogynium timoriense (Burdekin plum, seeds (BPS) and flesh (BPF)). The methanolic extracts of the plants samples were analysed for Total phenolic content (TPC) and antioxidant capacity measure by FRAP. The highest values were found in the KPF which were 12,442 ± 1355 mg GAE/ 100 g TPC and 16,670 ± 2275 mg TXE/100 g antioxidant capacity. Extracts of GGL was deemed to be most potent with complete cell inhibition in HeLa and HT29, and about 95% inhibition in HuH7 cells. Comparative activity was also seen for KPS extract, where more than 80% cell inhibition occurred in all tested cell lines. Dose-dependent studies showed higher SI values (0.72-1.02) in KPS extracts than GGL (0.5-0.73). Microbial assays of the crude extracts were also performed against five bacterial strains commonly associated with causing food poisoning diseases were selected (Gram positive-Staphylococcus aureus and Gram negative-Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa bacteria). KPF extracts were effective in suppressing microbial growth of all tested bacterial strains except for P. aeruginosa, while TKS and TKF were only slightly effective against S. aureus. Due to the potential of the GGL crude extract to completely inhibit the cells compared to KPS, it was further fractionated and tested against the cell lines. HPLC phenolic profiling of the crude extracts were performed, and numerous peak overlaps were evident in the fruit extracts. The KPF extracts demonstrated the strongest peaks which was coherent with the fact that it had the highest TPC and antioxidant capacity values. A high occurrence of t-ferulic acid in the GGL extracts was found which may explain the cytotoxic activity of GGL extracts. Peaks in KPS and KPF extracts were tentatively identified as gallic acid, protocatechuic acid, 4-hydroxybenzoic acid and syringic acid and possibly ellagic acid. HPLC time-based fractionation of the GGL extract (F1-F5) was performed and Dose dependent cytotoxic effects were determined. It was construed that F1, having the highest SI value for HeLa, HT29 and HuH7 (1.60, 1.41 and 1.67, respectively) would be promising for further fractionation and isolation process.

Genome-wide mapping of ZNF652 promoter binding sites in breast cancer cells.

A significant proportion of transcription factors encoded by the human genome are classical C(2) H(2) zinc finger proteins that regulate gene expression by directly interacting with their cognate DNA binding motifs. We previously showed that one such C(2) H(2) zinc finger DNA binding protein, ZNF652 (zinc finger protein 652), specifically and functionally interacts with CBFA2T3 to repress transcription of genes involved in breast oncogenesis. To identify potential targets by which ZNF652 exerts its putative tumour suppressive function, its promoter-specific cistrome was mapped by ChIP-chip. De novo motif scanning of the ZNF652 binding sites identified a novel ZNF652 recognition motif that closely resembles the previously characterised in vitro binding site, being a 10 nucleotide core of that 13 nucleotide sequence. Genes with ZNF652 binding sites function in diverse cellular pathways, and many are involved in cancer development and progression. Characterisation of the in vivo ZNF652 DNA binding motif and identification of potential ZNF652 target genes are key steps towards elucidating the function(s) of this transcription factor in the normal and malignant breast cell.

A PtdInsP(3)- and Rho GTPase-mediated positive feedback loop regulates neutrophil polarity.

When presented with a gradient of chemoattractant, many eukaryotic cells respond with polarized accumulation of the phospholipid PtdIns(3,4,5)P(3). This lipid asymmetry is one of the earliest readouts of polarity in neutrophils, Dictyostelium discoideum and fibroblasts. However, the mechanisms that regulate PtdInsP(3) polarization are not well understood. Using a cationic lipid shuttling system, we have delivered exogenous PtdInsP(3) to neutrophils. Exogenous PtdInsP(3) elicits accumulation of endogenous PtdInsP(3) in a positive feedback loop that requires endogenous phosphatidylinositol-3-OH kinases (PI(3)Ks) and Rho family GTPases. This feedback loop is important for establishing PtdInsP(3) polarity in response to both chemoattractant and to exogenous PtdInsP(3); it may function through a self-organizing pattern formation system. Emergent properties of positive and negative regulatory links between PtdInsP(3) and Rho family GTPases may constitute a broadly conserved module for the establishment of cell polarity during eukaryotic chemotaxis.

Targeting the p53 Pathway in Ewing Sarcoma.

The p53 tumour suppressor plays a pivotal role in the prevention of oncogenic transformation. Cancers frequently evade the potent antitumour surveillance mechanisms of p53 through mutation of the TP53 gene, with approximately 50% of all human malignancies expressing dysfunctional, mutated p53 proteins. Interestingly, genetic lesions in the TP53 gene are only observed in 10% of Ewing Sarcomas, with the majority of these sarcomas expressing a functional wild-type p53. In addition, the p53 downstream signaling pathways and DNA-damage cell cycle checkpoints remain functionally intact in these sarcomas. This paper summarizes recent insights into the functional capabilities and regulation of p53 in Ewing Sarcoma, with a particular focus on the cross-talk between p53 and the EWS-FLI1 gene rearrangement frequently associated with this disease. The development of several activators of p53 is discussed, with recent evidence demonstrating the potential of small molecule p53 activators as a promising systemic therapeutic approach for the treatment of Ewing Sarcomas with wild-type p53.

Nasopharyngeal carcinoma among the Bidayuh of Sarawak, Malaysia: History and risk factors.

Nasopharyngeal carcinoma (NPC) is a cancer of the epithelial cells lining the nasopharynx. The incidence of NPC has a distinct geographical distribution, mainly affecting the Chinese population of Southern China. In Malaysia, this cancer is exceptionally prevalent among males. There is a high incidence rate of NPC among the Bidayuh natives in Sarawak, Malaysia. Other than epidemiology reports, there has not been an article describing plausible cancer risk factors contributing to NPC within this native group. Researchers are still trying to understand the reasons the Bidayuh and Southern Chinese are highly susceptible to NPC. This article discusses the risk factors of developing NPC: Epstein-Barr virus infection, genetic predisposition, diet, environmental exposure and tobacco smoking. There is a need to improve the understanding of the role of risk factors to identify new ways to prevent cancer, especially among high-risk groups.

Antioxidative and therapeutic potential of selected Australian plants: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Numerous common pharmaceuticals, including anti-cancer, antiviral and antidiabetic drugs, are derived from traditional plant-derived medicines. With approximately 25,000 species of flora occurring in Australia that are adapted to the harsh environment, there is a plethora of novel compounds awaiting research in the context of their medicinal properties. Anecdotal accounts of plant-based medicines used by the Australian Aboriginal and Torres Strait Islander peoples clearly illustrates high therapeutic activity. AIM: This review aims to demonstrate the medicinal potentials of selected native Australian plants based on scientific data. Furthermore, it is anticipated that work presented here will contribute towards enhancing our knowledge of native plants from Australia, particularly in the prevention and potential treatment of disease types such as cancer, microbial and viral infections, and diabetes. This is not meant to be a comprehensive study, rather it is meant as an overview to stimulate future research in this field. METHODS: The EBSCOhost platform which included PubMed, SciFinder, Web of Knowledge, Scopus, and ScienceDirect databases were searched for papers using the keywords: medicinal plants, antioxidative, antimicrobial, antibacterial, anticancer, anti-tumor, antiviral or antidiabetic, as well as Australian, native, traditional and plants. The selection criteria for including studies were restricted to articles on plants used in traditional remedies which showed antioxidative potential and therapeutic properties such as anticancer, antimicrobial, antiviral and antidiabetic activity. RESULTS: Some plants identified in this review which showed high Total Phenolic Content (TPC) and antioxidative capacity, and hence prominent bioactivity, included Tasmannia lanceolata (Poir.) A.C. Sm., Terminalia ferdinandiana Exell, Eucalyptus species, Syzygium species, Backhousia citriodora F.Muell., Petalostigma species, Acacia species, Melaleuca alternifolia (Maiden & Betche) Cheel, Eremophila species, Prostanthera rotundifolia R.Br., Scaevola spinescens R. Br. and Pittosporum angustifolium Lodd. The majority of studies found polar compounds such as caffeic acid, coumaric acid, chlorogenic acid, quercetin, anthocyanins, hesperidin, kaempferol, catechin, ellagic acid and saponins to be the active components responsible for the therapeutic effects. Additionally, mid to non-polar volatile organic compounds such as meroterpenes (serrulatanes and nerol cinnamates), monoterpenes (1,8-cineole and myodesert-1-ene), sesquiterpenes, diterpenes and triterpenes, that are known only in Australian plants, have also shown therapeutic properties related to traditional medicine. CONCLUSION: Australian plants express a diverse range of previously undescribed metabolites that have not been given full in vitro assessment for human health potential. This review has included a limited number of plant species of ethnomedicinal significance; hundreds of plants remain in need of exploration and detailed study. Future more elaborate studies are therefore required to screen out and purify lead bioactive compounds against numerous other disease types. This will not only improve our knowledge on the phytochemistry of Australian native flora, but also provide a platform to understand their health-promoting and bioactive effects for pharmaceutical interventions, nutraceuticals, cosmetics, and as functional foods. Finally, plant-derived natural compounds (phytochemicals), as well as plant-based traditional remedies, are significant sources for latent and novel drugs against diseases. Extensive investigation of native medicinal plants may well hold the key to novel drug discoveries.

P53 activation suppresses irinotecan metabolite SN-38-induced cell damage in non-malignant but not malignant epithelial colonic cells.

Nutlin-3a is a p53 activator and potential cyclotherapy approach that may also mitigate side effects of chemotherapeutic drugs in the treatment of colorectal cancer. We investigated cell proliferation in a panel of colorectal cancer (CRC) cell lines with wild-type or mutant p53, as well as a non-tumorigenic fetal intestinal cell line following Nutlin-3a treatment (10 µM). We then assessed apoptosis at 24 and 48 h following administration of the active irinotecan metabolite, SN-38 (0.001 µM - 1 µM), alone or following pre-treatment with Nutlin-3a (10 µM). Nutlin-3a treatment (10 µM) significantly reduced proliferation in wild-type p53 expressing cell lines (FHS 74 and HCT116+/+) at 72 and 96 h, but was without effect in cell lines with mutated or deleted p53 (Caco-2, SW480, and HCT 116-/-). SN-38 treatment induced significant apoptosis in all cell lines after 48 h. Nutlin-3a unexpectedly increased cell death in the p53 wild-type CRC cell line, HCT116+/+, while Nutlin-3a pre-treatment provided protection from SN-38 in the p53 wild-type normal cell line, FHs 74. These results demonstrate Nutlin-3a's selective growth-arresting efficacy in p53 wild-type non-malignant intestinal cell lines, enabling the selective targeting of malignant cells with chemotherapy drugs. These studies highlight the potential of Nutlin-3a to minimise intestinal mucosal damage following chemotherapy.

Quantifying human post-mortem movement resultant from decomposition processes.

BACKGROUND: Post-mortem movement is highly significant in unexplained death investigations, as body position or the position of remains helps to determine cause and manner of death, as well as potentially the circumstances surrounding death. Therefore, understanding post-mortem movement is of forensic relevance in death scene assessments. PURPOSE: The aim of this study was to quantify post-mortem movement in anatomical structures of a human donor during decomposition in an Australian environment, an evaluation that has not previously been undertaken. METHODS: The aim was achieved using time-lapse images of a human donor decomposing in order to capture the post-mortem movement over a 16-month period. Megyesi et al.'s [1] total body score system was used to quantify the decomposition of the donor in each image to determine the decomposition stage. ImageJ software was used to determine the distance from static landmarks to anatomical structures of interest in each image to allow for quantification. RESULTS: Early decomposition progressed rapidly, and advanced decomposition plateaued at 41 post-mortem interval days with a total body score of 24. The results support the conclusion that post-mortem movement does occur in all limbs of the donor. The anatomical structure that produced the most movement was the right styloid process of the radius, moving a total distance of 51.65 cm. A surprising finding of the study was that most post-mortem movement occurs in the advanced decomposition stage, with the lower limbs being the most active. CONCLUSION: This study supports that post-mortem movement can be quantified using time-lapse imagery, with results supporting movement in all limbs, a process that was active for the entire study period. An interesting finding was that the decomposition plateaued in the advanced stage with the donor remaining in mummification, and not reaching skeletonization after 16 months in situ. These findings are of significant importance to police in death scene assessments and forensic investigations.

Natural product-derived phytochemicals as potential agents against coronaviruses: A review.

Coronaviruses are responsible for a growing economic, social and mortality burden, as the causative agent of diseases such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), avian infectious bronchitis virus (IBV) and COVID-19. However, there is a lack of effective antiviral agents for many coronavirus strains. Naturally existing compounds provide a wealth of chemical diversity, including antiviral activity, and thus may have utility as therapeutic agents against coronaviral infections. The PubMed database was searched for papers including the keywords coronavirus, SARS or MERS, as well as traditional medicine, herbal, remedy or plants, with 55 primary research articles identified. The overwhelming majority of publications focussed on polar compounds. Compounds that show promise for the inhibition of coronavirus in humans include scutellarein, silvestrol, tryptanthrin, saikosaponin B2, quercetin, myricetin, caffeic acid, psoralidin, isobavachalcone, and lectins such as griffithsin. Other compounds such as lycorine may be suitable if a therapeutic level of antiviral activity can be achieved without exceeding toxic plasma concentrations. It was noted that the most promising small molecules identified as coronavirus inhibitors contained a conjugated fused ring structure with the majority being classified as being polyphenols.

In vitro cytotoxicity evaluation of thiourea derivatives bearing Salix sp. constituent against HK-1 cell lines.

In searching for drugs from natural product scaffolds has gained interest among researchers. In this study, a series of twelve halogenated thiourea (ATX 1-12) via chemical modification of aspirin (a natural product derivative) and evaluated for cytotoxic activity against nasopharyngeal carcinoma (NPC) cell lines, HK-1 via MTS-based colorimetric assay. The cytotoxicity studies demonstrated that halogens at meta position of ATX showed promising activity against HK-1 cells (IC50 value ≤15 µM) in comparison to cisplatin, a positive cytotoxic drug (IC50 value =8.9 ± 1.9 µM). ATX 11, bearing iodine at meta position, showed robust cytotoxicity against HK-1 cells with an IC50 value of 4.7 ± 0.7 µM. Molecular docking interactions between ATX 11 and cyclooxygenase-2 demonstrated a robust binding affinity value of -8.1 kcal/mol as compared to aspirin's binding affinity value of -6.4 kcal/mol. The findings represent a promising lead molecule from natural product with excellent cytotoxic activity against NPC cell lines.

Quantification of PtdIns(3,4,5)P(3) dynamics in EGF-stimulated carcinoma cells: a comparison of PH-domain-mediated methods with immunological methods.

Class IA PI3Ks (phosphoinositide 3-kinases) generate the secondary messenger PtdIns(3,4,5)P(3), which plays an important role in many cellular responses. The accumulation of PtdIns(3,4,5)P(3) in cell membranes is routinely measured using GFP (green fluorescent protein)-labelled PH (pleckstrin homology) domains. However, the kinetics of membrane PtdIns(3,4,5)P(3) synthesis and turnover as detected by PH domains have not been validated using an independent method. In the present study, we measured EGF (epidermal growth factor)-stimulated membrane PtdIns(3,4,5)P(3) production using a specific monoclonal anti-PtdIns(3,4,5)P(3) antibody, and compared the results with those obtained using PH-domain-dependent methods. Anti-PtdIns(3,4,5)P(3) staining rapidly accumulated at the leading edge of EGF-stimulated carcinoma cells. PtdIns(3,4,5)P(3) levels were maximal at 1 min, and returned to basal levels by 5 min. In contrast, membrane PtdIns(3,4,5)P(3) production, measured by the membrane translocation of an epitope-tagged (BTK)PH (PH domain of Bruton's tyrosine kinase), remained approx. 2-fold above basal level throughout 4-5 min of EGF stimulation. To determine the reason for this disparity, we measured the rate of PtdIns(3,4,5)P(3) hydrolysis by measuring the decay of the PtdIns(3,4,5)P(3) signal after LY294002 treatment of EGF-stimulated cells. LY294002 abolished anti-PtdIns(3,4,5)P(3) membrane staining within 10 s of treatment, suggesting that PtdIns(3,4,5)P(3) turnover occurs within seconds of synthesis. In contrast, (BTK)PH membrane recruitment, once initiated by EGF, was relatively insensitive to LY294002. These data suggest that sequestration of PtdIns(3,4,5)P(3) by PH domains may affect the apparent kinetics of PtdIns(3,4,5)P(3) accumulation and turnover; consistent with this hypothesis, we found that GRP-1 (general receptor for phosphoinositides 1) PH domains [which, like BTK, are specific for PtdIns(3,4,5)P(3)] inhibit PTEN (phosphatase and tensin homologue deleted on chromosome 10) dephosphorylation of PtdIns(3,4,5)P(3) in vitro. These data suggest that anti-PtdIns(3,4,5)P(3) antibodies are a useful tool to detect localized PtdIns(3,4,5)P(3), and illustrate the importance of using multiple approaches for the estimation of membrane phosphoinositides.

FBXO31 is the chromosome 16q24.3 senescence gene, a candidate breast tumor suppressor, and a component of an SCF complex.

A BAC located in the 16q24.3 breast cancer loss of heterozygosity region was previously shown to restore cellular senescence when transferred into breast tumor cell lines. We have shown that FBXO31, although located just distal to this BAC, can induce cellular senescence in the breast cancer cell line MCF-7 and is the likely candidate senescence gene. FBXO31 has properties consistent with a tumor suppressor, because ectopic expression of FBXO31 in two breast cancer cell lines inhibited colony growth on plastic and inhibited cell proliferation in the MCF-7 cell line. In addition, compared with the relative expression in normal breast, levels of FBXO31 were down-regulated in breast tumor cell lines and primary tumors. FBXO31 was cell cycle regulated in the breast cell lines MCF-10A and SKBR3 with maximal expression from late G(2) to early G(1) phase. Ectopic expression of FBXO31 in the breast cancer cell line MDA-MB-468 resulted in the accumulation of cells at the G(1) phase of the cell cycle. FBXO31 contains an F-box domain and is associated with the proteins Skp1, Roc-1, and Cullin-1, suggesting that FBXO31 is a component of a SCF ubiquitination complex. We propose that FBXO31 functions as a tumor suppressor by generating SCF(FBXO31) complexes that target particular substrates, critical for the normal execution of the cell cycle, for ubiquitination and subsequent degradation.

Cellular calcium mobilization in response to phosphoinositide delivery.

Intracellular calcium [Ca(2+)](i) is mobilized in many cell types in response to activation of phosphoinositide (PIP(n)) signaling pathways involving PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3). To further explore the relationship between increases in intracellular PIP(n) concentrations and mobilization of [Ca(2+)](i), each of the seven phosphorylated phosphoinositides (PIP(n)s) were delivered into cells and the metabolism and physiological effects of the exogenously administered PIP(n)s were determined. The efficient cellular delivery of fluorophore-tagged and native PIP(n)s was accomplished using histone protein, neomycin, and dendrimeric polyamines. PtdIns(4,5)P(2) fluorophore-tagged analogs with short- and long-acyl chains were substrates for cellular enzymes in vitro and for phospholipases in stimulated fibroblasts. PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2), each induced calcium mobilization rapidly after exogenous addition to fibroblasts. PtdIns(3,4,5)P(3) induced a significant, but smaller increase in intracellular calcium. These observations suggest that PIP(n)s other than PtdIns(4,5)P(2) or PtdIns(3,4,5)P(3) may have direct roles in signaling involving [Ca(2+)](i).

p53 Represses the Oncogenic Sno-MiR-28 Derived from a SnoRNA.

p53 is a master tumour repressor that participates in vast regulatory networks, including feedback loops involving microRNAs (miRNAs) that regulate p53 and that themselves are direct p53 transcriptional targets. We show here that a group of polycistronic miRNA-like non-coding RNAs derived from small nucleolar RNAs (sno-miRNAs) are transcriptionally repressed by p53 through their host gene, SNHG1. The most abundant of these, sno-miR-28, directly targets the p53-stabilizing gene, TAF9B. Collectively, p53, SNHG1, sno-miR-28 and TAF9B form a regulatory loop which affects p53 stability and downstream p53-regulated pathways. In addition, SNHG1, SNORD28 and sno-miR-28 are all significantly upregulated in breast tumours and the overexpression of sno-miR-28 promotes breast epithelial cell proliferation. This research has broadened our knowledge of the crosstalk between small non-coding RNA pathways and roles of sno-miRNAs in p53 regulation.