Shifts in macrophyte species composition as a result of eutrophication and pollution in Dutch transboundary streams over the past decades

The catchment areas of transboundary streams in the Netherlands have been subject to increasing agricultural and industrial activities over the past decades. To evaluate the effects of these activities on the aquatic vegetation, a study has been carried out in 28 Dutch transboundary lowland streams. Recent data on distribution of 58 aquatic plant species and their growth forms were compared with historical data and were correlated with abiotic variables. Most of these streams lost species that are characteristic for streams and are sensitive to turbidity, eutrophication and pollution (e.g. Potamogeton alpinus, P. polygonifolius, P. densus, Ranunculus peltatus ssp. heterophyllus, Callitriche stagnalis and Myriophyllum alterniflorum.) Species, not common in streams but tolerant to turbidity, eutrophication or pollution (e.g. Potamogeton trichoides, Elodea nuttallii) appeared in many streams or increased in abundance. There was also a shift in growth forms: submerged species decreased or were replaced by emergent/floating-leaved species. Correspondence analysis was carried out to study the relation between the observed changes and the abiotic characteristic of the streams. The magnitude of the shift in species composition was positively correlated with the PO₄ ^3- concentration and pH (which was highly correlated with Cd²⁺) of the water. This leads to the hypothesis that increased input of sewage, agricultural and industrial water causes a change in species composition and main growth forms of aquatic plant species in lowland streams.     

Inhibition of DNA-dependent protein kinase induces accelerated senescence in irradiated human cancer cells

DNA-dependent protein kinase (DNA-PK) plays a pivotal role in the repair of DNA double-strand breaks (DSB) and is centrally involved in regulating cellular radiosensitivity. Here, we identify DNA-PK as a key therapeutic target for augmenting accelerated senescence in irradiated human cancer cells. We find that BEZ235, a novel inhibitor of DNA-PK and phosphoinositide 3-kinase (PI3K)/mTOR, abrogates radiation-induced DSB repair resulting in cellular radiosensitization and growth delay of irradiated tumor xenografts. Importantly, radiation enhancement by BEZ235 coincides with a prominent p53-dependent accelerated senescence phenotype characterized by positive β-galactosidase staining, G(2)-M cell-cycle arrest, enlarged and flattened cellular morphology, and increased p21 expression and senescence-associated cytokine secretion. Because this senescence response to BEZ235 is accompanied by unrepaired DNA DSBs, we examined whether selective targeting of DNA-PK also induces accelerated senescence in irradiated cells. Significantly, we show that specific pharmacologic inhibition of DNA-PK, but not PI3K or mTORC1, delays DSB repair leading to accelerated senescence after radiation. We additionally show that PRKDC knockdown using siRNA promotes a striking accelerated senescence phenotype in irradiated cells comparable with that of BEZ235. Thus, in the context of radiation treatment, our data indicate that inhibition of DNA-PK is sufficient for the induction of accelerated senescence. These results validate DNA-PK as an important therapeutic target in irradiated cancer cells and establish accelerated senescence as a novel mechanism of radiosensitization induced by DNA-PK blockade.     

RNAi-mediated knock-down of arylamine N-acetyltransferase-1 expression induces E-cadherin up-regulation and cell-cell contact growth inhibition

Arylamine N-acetyltransferase-1 (NAT1) is an enzyme that catalyzes the biotransformation of arylamine and hydrazine substrates. It also has a role in the catabolism of the folate metabolite p-aminobenzoyl glutamate. Recent bioinformatics studies have correlated NAT1 expression with various cancer subtypes. However, a direct role for NAT1 in cell biology has not been established. In this study, we have knocked down NAT1 in the colon adenocarcinoma cell-line HT-29 and found a marked change in cell morphology that was accompanied by an increase in cell-cell contact growth inhibition and a loss of cell viability at confluence. NAT1 knock-down also led to attenuation in anchorage independent growth in soft agar. Loss of NAT1 led to the up-regulation of E-cadherin mRNA and protein levels. This change in E-cadherin was not attributed to RNAi off-target effects and was also observed in the prostate cancer cell-line 22Rv1. In vivo, NAT1 knock-down cells grew with a longer doubling time compared to cells stably transfected with a scrambled RNAi or to parental HT-29 cells. This study has shown that NAT1 affects cell growth and morphology. In addition, it suggests that NAT1 may be a novel drug target for cancer therapeutics.     

Amplicon-dependent CCNE1 expression is critical for clonogenic survival after cisplatin treatment and is correlated with 20q11 gain in ovarian cancer

Genomic amplification of 19q12 occurs in several cancer types including ovarian cancer where it is associated with primary treatment failure. We systematically attenuated expression of genes within the minimally defined 19q12 region in ovarian cell lines using short-interfering RNAs (siRNA) to identify driver oncogene(s) within the amplicon. Knockdown of CCNE1 resulted in G1/S phase arrest, reduced cell viability and apoptosis only in amplification-carrying cells. Although CCNE1 knockdown increased cisplatin resistance in short-term assays, clonogenic survival was inhibited after treatment. Gain of 20q11 was highly correlated with 19q12 amplification and spanned a 2.5 Mb region including TPX2, a centromeric protein required for mitotic spindle function. Expression of TPX2 was highly correlated with gene amplification and with CCNE1 expression in primary tumors. siRNA inhibition of TPX2 reduced cell viability but this effect was not amplicon-dependent. These findings demonstrate that CCNE1 is a key driver in the 19q12 amplicon required for survival and clonogenicity in cells with locus amplification. Co-amplification at 19q12 and 20q11 implies the presence of a cooperative mutational network. These observations have implications for the application of targeted therapies in CCNE1 dependent ovarian cancers.     

Angiopoietin-2 Stimulation of Endothelial Cells Induces αvβ3 Integrin Internalization and Degradation

The angiopoietins (Ang-1 and Ang-2) have been identified as agonistic and antagonistic ligands of the endothelial receptor tyrosine kinase Tie2, respectively. Both ligands have been demonstrated to induce translocation of Tie2 to cell-cell junctions. However, only Ang-1 induces Tie2-dependent Akt activation and subsequent survival signaling and endothelial quiescence. Ang-2 interferes negatively with Ang-1/Tie2 signaling, thereby antagonizing the Ang-1/Tie2 axis. Here, we show that both Ang-1 and Ang-2 recruit β3 integrins to Tie2. This co-localization is most prominent in cell-cell junctions. However, only Ang-2 stimulation resulted in complex formation among Tie2, αvβ3 integrin, and focal adhesion kinase as evidenced by co-immunoprecipitation experiments. Focal adhesion kinase was phosphorylated in the FAT domain at Ser⁹¹⁰ upon Ang-2 stimulation and the adaptor proteins p130Cas and talin dissociated from αvβ3 integrin. The αvβ3 integrin was internalized, ubiquitinylated, and gated toward lysosomes. Taken together, the experiments define Tie2/αvβ3 integrin association-induced integrin internalization and degradation as mechanistic consequences of endothelial Ang-2 stimulation.     

Crystal structure of a dimeric form of streptococcal pyrogenic exotoxin A (SpeA1)

Streptococcal pyrogenic exotoxin A (SpeA1) is a bacterial superantigen associated with scarlet fever and streptococcal toxic shock syndrome (STSS). SpeA1 is found in both monomeric and dimeric forms, and previous work suggested that the dimer results from an intermolecular disulfide bond between the cysteines at positions 90 of each monomer. Here, we present the crystal structure of the dimeric form of SpeA1. The toxin crystallizes in the orthorhombic space group P212121, with two dimers in the crystallographic asymmetric unit. The final structure has a crystallographic R-factor of 21.52% for 7248 protein atoms, 136 water molecules, and 4 zinc atoms (one zinc atom per molecule). The implications of SpeA1 dimer on MHC class II and T-cell receptor recognition are discussed.     

Preparation and cell growth inhibitory activity of [PtR(2)L(2)] (R=polyfluorophenyl,L(2)=diene,cyclohexane-1,2-diamine (chxn) or cis-(dimethyl sulfoxide)(2)) and the X-ray crystal structure of [Pt(C(6)F(5))(2)(cis-chxn)

A range of [PtR(2)(chxn)] (R=C(6)F(5), o-HC(6)F(4), p-HC(6)F(4), p-MeOC(6)F(4) or 3,5-H(2)C(6)F(3); chxn=cyclohexane-1,2-diamine) and cis-[PtR(2)(dmso)(2)] (R=C(6)F(5), p-HC(6)F(4) or p-MeOC(6)F(4); dmso=dimethyl sulfoxide) complexes have been prepared from the corresponding [PtR(2)(diene)] (diene=cis,cis-cycloocta-1,5-diene (cod), hexa-1,5-diene (hex), norbornadiene (nbd) or dicyclopentadiene (dcy)) derivatives and have been spectroscopically characterized. A representative crystal structure of [Pt(C(6)F(5))(2)(cis-chxn)] was determined and shows a slightly distorted square planar geometry for platinum with chxn virtually perpendicular to the coordination plane. The biological activity against L1210 and L1210/DDP cell lines of these compounds together with the behaviour of other organoplatinum complexes, [PtR(2)L(2)] (L(2)=ethane-1,2-diamine (en) or cis-(NH(3))(2)) have been determined. Despite the use of relatively inert fluorocarbon anions as leaving groups, moderate-high cell growth inhibitory activity is observed. None of the fluorocarbon complexes displayed any cross resistance with cisplatin.