Identification and functional characterization of a human GalNAc [alpha]2,6-sialyltransferase with altered expression in breast cancer

BACKGROUND: We sought to identify genes with altered expression during human breast cancer progression by applying mRNA comparisons of normal and tumor mammary cell lines with increasingly malignant phenotypes. The gene encoding a new sialyltransferase (STM) was found to be down-regulated in tumor cells. Abnormal expression and enzymatic activities of sialyltransferases in tumor cells result in the formation of tumor-associated carbohydrate antigens that can be used for the better understanding of the disease process and are applied for tumor diagnosis and immunotherapy. Altered glycosylation patterns of the MUC1 mucin, in particular, is a target antigen for immunotherapy of breast and other cancers. MATERIALS AND METHODS: Total RNAs from multiple normal mammary epithelial cell strains and tumor cell lines were compared by differential display and the differential expression of selected cDNAs was confirmed by Northern analyses. Recombinant STM was expressed in COS-7 cells. The substrate and linkage specificity of STM was examined using various oligosaccharides and O-glycosylated proteins as acceptor substrates. The chromosomal localization of the SIATL1 gene was assigned by somatic cell hybrid analysis. RESULTS: A human sialyltransferase gene was identified by differential display as being down-regulated in breast tumor cell lines as compared to normal mammary epithelial cell strains, and the corresponding full-length cDNA (stm) was cloned. The encoded protein of 374 amino acid residues contained the L- and S-sialylmotifs, two catalytic regions conserved in all functional sialyltransferases. Recombinant STM is an active GalNAc alpha2,6-sialyltransferase with Gal beta 1,3 GalNAc-O-Ser/Thr and (+/- Neu5Ac alpha 2,3) Gal beta 1,3GalNAc-O-Ser/Thr acceptor specificity. The SIATL1 gene, encoding STM, was mapped to the long arm of human chromosome 17 at q23-qter, a region that is nonrandomly deleted in human breast cancers. However, Southern analyses indicated that SIATL1 is usually not grossly rearranged in breast tumors. Northern analyses showed that the gene was widely expressed in normal human tissues, as well as in normal breast and prostate epithelial cell lines, but significantly down-regulated or absent in corresponding tumor cell lines. CONCLUSIONS: Our findings suggest that aberrant expression of STM sialyltransferase in tumors could be a feature of the malignant phenotype. In breast cancers, the MUC1 mucin is overexpressed and contains shorter O-glycans as compared to the normal mucin. Because STM catalyzes the synthesis of O-glycans, cloning and characterization of its substrate specificity will contribute to the understanding of the molecular mechanisms underlying the aberrant glycosylation patterns of O-glycans and the formation of mucin-related antigens in human breast cancers.     

CYP3A4-V and prostate cancer in African Americans: causal or confounding association because of population stratification?

CYP3A4-V, an A to G promoter variant associated with prostate cancer in African Americans, exhibits large differences in allele frequency between populations. Given that the African American population is genetically heterogeneous because of its African ancestry and subsequent admixture with European Americans, case-control studies with African Americans are highly susceptible to spurious associations. To test for association with prostate cancer, we genotyped CYP3A4-V in 1376 (2 N) chromosomes from prostate cancer patients and age- and ethnicity-matched controls representing African Americans, Nigerians, and European Americans. To detect population stratification among the African American samples, 10 unlinked genetic markers were genotyped. To correct for the stratification, the uncorrected association statistic was divided by the average of association statistics across the 10 unlinked markers. Sharp differences in CYP3A4-V frequencies were observed between Nigerian and European American controls (0.87 and 0.10, respectively; P<0.0001). African Americans were intermediate (0.66). An association uncorrected for stratification was observed between CYP3A4-V and prostate cancer in African Americans (P=0.007). A nominal association was also observed among European Americans (P=0.02) but not Nigerians. In addition, the unlinked genetic marker test provided strong evidence of population stratification among African Americans. Because of the high level of stratification, the corrected P-value was not significant (P=0.25). Follow-up studies on a larger dataset will be needed to confirm whether the association is indeed spurious; however, these results reveal the potential for confounding of association studies by using African Americans and the need for study designs that take into account substructure caused by differences in ancestral proportions between cases and controls.     

Proteolytic activity against the light-harvesting complex and the D1/D2 core proteins of Photosystem II in close association to the light-harvesting complex II trimer

Light-harvesting complex II (LHCII) prepared from isolated thylakoids of either broken or intact chloroplasts by three independent methods, exhibits proteolytic activity against LHCII. This activity is readily detectable upon incubation of these preparations at 37 degrees C (without addition of any chemicals or prior pre-treatment), and can be monitored either by the LHCII immunostain reduction on Western blots or by the Coomassie blue stain reduction in substrate-containing "activity gels". Upon SDS-sucrose density gradient ultracentrifugation of SDS-solubilized thylakoids, a method which succeeds in the separation of the pigment-protein complexes in their trimeric and monomeric forms, the protease activity copurifies with the LHCII trimer, its monomer exhibiting no activity. This LHCII trimer, apart from being "self-digested", also degrades the Photosystem II (PSII) core proteins (D1, D2) when added to an isolated PSII core protein preparation containing the D1/D2 heterodimer. Under our experimental conditions, 50% of LHCII or the D1, D2 proteins are degraded by the LHCII-protease complex within 30 min at 37 degrees C and specific degradation products are observed. The protease is light-inducible during chloroplast biogenesis, stable in low concentrations of SDS, activated by Mg(2+), and inhibited by Zn(2+), Cd(2+), EDTA and p-hydroxy-mercury benzoate (pOHMB), suggesting that it may belong to the cysteine family of proteases. Upon electrophoresis of the LHCII trimer on substrate-containing "activity gels" or normal Laemmli gels, the protease is released from the complex and runs in the upper part of the gel, above the LHCII trimer. A polypeptide of 140 kDa that exhibits proteolytic activity against LHCII, D1 and D2 has been identified as the protease. We believe that this membrane-bound protease is closely associated to the LHCII complex in vivo, as an LHCII-protease complex, its function being the regulation of the PSII unit assembly and/or adaptation.     

Triazines facilitate neurotransmitter release of synaptic terminals located in hearts of frog (Rana ridibunda) and honeybee (Apis mellifera) and in the ventral nerve cord of a beetle (Tenebrio molitor)

Three triazine herbicides, atrazine, simazine and metribuzine, and some of their major metabolites (cyanuric acid and 6-azauracil) were investigated for their action on synaptic terminals using three different isolated tissue preparations from the atria of the frog, Rana ridibunda, the heart of the honeybee, Apis mellifera macedonica, and the ventral nerve cord of the beetle, Tenebrio molitor. The results indicate that triazines facilitate the release of neurotransmitters from nerve terminals, as already reported for the mammalian central nervous system. The no observed effect concentration, the maximum concentration of the herbicide diluted in the saline that has no effect on the physiological properties of the isolated tissue, was estimated for each individual preparation. According to their relative potency, the three triazines tested can be ranked as follows: atrazine (cyanuric acid), simazine>metribuzine (6-azauracil). The action of these compounds on the cholinergic (amphibians, insects), adrenergic (amphibian) and octopaminergic (insects) synaptic terminals is discussed.     

The simultaneous determination of 1-aminocyclopropane-1-carboxylic acid and cyclopropane-1,1-dicarboxylic acid in Lycopersicum esculentum by high-performance liquid chromatography--electrospray tandem mass spectrometry

Varying concentrations of cyclopropane-1,1-dicarboxylic acid (CDA), an inhibitor of 1-aminocyclopropane-1-carboxylic acid oxidase, added to the solid culture medium of tomato nodal shoot segments resulted in a reduction in the level of endogenous ethylene according to the concentration of inhibitor applied. Following treatment with inhibitor, plants were homogenised and the concentrations of CDA and of 1-aminocyclopropane-1-carboxylic acid (ACC) were measured simultaneously in the resulting juice using an HPLC-ESI/MS-MS method. The levels of CDA and ACC measured in the plant tissues were associated with the concentration of inhibitor added to the solid medium. The HPLC-ESI/MS-MS method described produced limits of detection of 0.8 pmol for ACC and of 4 pmol for CDA.     

Origin and function of tumor stroma fibroblasts

Tumor development is critically dependent on the formation of a supporting stroma consisting of neovasculature, inflammatory cells and activated fibroblasts. Activated fibroblasts present a heterogeneous cell population not only in regard to the expression of marker molecules but also to their origin and molecular signaling properties. The plasticity of this cell type is pointed out by the multiple transdifferentiation events that lead to the generation of activated fibroblasts which can arise from resting fibroblasts, epithelial and endothelial cells as well as from mesenchymal stem cells. Cellular in vitro and in vivo experiments have changed the perspective of fibroblasts from passive “bystanders” in the tumor microenvironment to that of important drivers of tumor progression. Here, we describe the multiple origins of fibroblast recruitment to the tumor tissue as well as the function of activated fibroblasts during tumor initiation, progression, metastasis and anti-VEGF resistance. The identification of markers present in activated fibroblasts as well as a better understanding how these cells influence other tumor compartments has led to the clinical development of anti-tumor therapies.     

Mechanisms of Basin-Scale Nitrogen Load Reductions under Intensified Irrigated Agriculture

Irrigated agriculture can modify the cycling and transport of nitrogen (N), due to associated water diversions, water losses, and changes in transport flow-paths. We investigate dominant processes behind observed long-term changes in dissolved inorganic nitrogen (DIN) concentrations and loads of the extensive (465,000 km2) semi-arid Amu Darya River basin (ADRB) in Central Asia. We specifically considered a 40-year period (1960–2000) of large irrigation expansion, reduced river water flows, increased fertilizer application and net increase of N input into the soil-water system. Results showed that observed decreases in riverine DIN concentration near the Aral Sea outlet of ADRB primarily were due to increased recirculation of irrigation water, which extends the flow-path lengths and enhances N attenuation. The observed DIN concentrations matched a developed analytical relation between concentration attenuation and recirculation ratio, showing that a fourfold increase in basin-scale recirculation can increase DIN attenuation from 85 to 99%. Such effects have previously only been observed at small scales, in laboratory experiments and at individual agricultural plots. These results imply that increased recirculation can have contributed to observed increases in N attenuation in agriculturally dominated drainage basins in different parts of the world. Additionally, it can be important for basin scale attenuation of other pollutants, including phosphorous, metals and organic matter. A six-fold lower DIN export from ADRB during the period 1981–2000, compared to the period 1960–1980, was due to the combined result of drastic river flow reduction of almost 70%, and decreased DIN concentrations at the basin outlet. Several arid and semi-arid regions around the world are projected to undergo similar reductions in discharge as the ADRB due to climate change and agricultural intensification, and may therefore undergo comparable shifts in DIN export as shown here for the ADRB. For example, projected future increases of irrigation water withdrawals between 2005 and 2050 may decrease the DIN export from arid world regions by 40%.     

Immune Response Gene Expression in Colorectal Cancer Carries Distinct Prognostic Implications According to Tissue,Stage and Site: A Prospective Retrospective Translational Study in the Context of a Hellenic Cooperative Oncology Group Randomised Trial

Background

Although host immune response is an emerging prognostic factor for colorectal cancer, there is no consensus on the optimal methodology, surrogate markers or tissue for study.

Patients and Methods

Tumour blocks were prospectively collected from 344 patients with stage II/III colorectal cancer (CRC) treated with adjuvant chemotherapy. Whole section lymphocytic infiltration was studied along with mRNA expression of CD3Z, CD8, CD4, CXCL9, CXCL13, IGHM, FOXP3, SNAI2 and ESR1 by qRT-qPCR in tissue microarray (TMA) cores from the centre of tumour, invasive margin and adjacent normal mucosa.

Results

Lymphocytic infiltration, deficient MMR (10.9%), KRAS (40.7%) and BRAF (4.9%) mutations or single mRNA gene expression were not prognostic. Tumour ESR1 gene expression (Hazard Ratio [HR] for relapse 2.33, 95% CI 1.35-4.02; HR for death 1.74, 95% CI 1.02-2.97) and absence of necrosis (HR for relapse 1.71, 95% CI 1.05-2.71; HR for death 1.98, 95% CI 1.14-3.43) were adverse prognostic features. We used CD3Z and CD8 expression in order to devise the mRNA-based Immune Score (mIS) and proceeded to partitioning analysis in 267 patients, with age, stage, tumour site (Right vs Left CRC), KRAS mutation and tumour mIS as input factors. Only in patients with stage III right-sided colon cancer, a low immune response was associated with inferior disease-free survival (mIS-low, HR for relapse 2.28, 95% CI 1.05-8.02). No prognostic significance was seen for tumour mIS in any other stage or site of CRC, or for a similar mIS score derived from adjacent normal mucosa. Independent adverse prognostic significance was retained in multivariable analysis for absence of necrosis, tumour ESR1 expression in all patients and low tumour mIS in stage III right-sided CRC.

Conclusions

In localised CRC, mRNA-based CD3Z/CD8 profiling of tumour immune response may have stage, site and tissue-specific prognostic significance, along with ESR1 expression.

Trial Registration

ANZCTR.org.au ACTRN12610000509066     

Pain Processing after Social Exclusion and Its Relation to Rejection Sensitivity in Borderline Personality Disorder

Objective

There is a general agreement that physical pain serves as an alarm signal for the prevention of and reaction to physical harm. It has recently been hypothesized that “social pain,” as induced by social rejection or abandonment, may rely on comparable, phylogenetically old brain structures. As plausible as this theory may sound, scientific evidence for this idea is sparse. This study therefore attempts to link both types of pain directly. We studied patients with borderline personality disorder (BPD) because BPD is characterized by opposing alterations in physical and social pain; hyposensitivity to physical pain is associated with hypersensitivity to social pain, as indicated by an enhanced rejection sensitivity.

Method

Twenty unmedicated female BPD patients and 20 healthy participants (HC, matched for age and education) played a virtual ball-tossing game (cyberball), with the conditions for exclusion, inclusion, and a control condition with predefined game rules. Each cyberball block was followed by a temperature stimulus (with a subjective pain intensity of 60% in half the cases). The cerebral responses were measured by functional magnetic resonance imaging. The Adult Rejection Sensitivity Questionnaire was used to assess rejection sensitivity.

Results

Higher temperature heat stimuli had to be applied to BPD patients relative to HCs to reach a comparable subjective experience of painfulness in both groups, which suggested a general hyposensitivity to pain in BPD patients. Social exclusion led to a subjectively reported hypersensitivity to physical pain in both groups that was accompanied by an enhanced activation in the anterior insula and the thalamus. In BPD, physical pain processing after exclusion was additionally linked to enhanced posterior insula activation. After inclusion, BPD patients showed reduced amygdala activation during pain in comparison with HC. In BPD patients, higher rejection sensitivity was associated with lower activation differences during pain processing following social exclusion and inclusion in the insula and in the amygdala.

Discussion

Despite the similar behavioral effects in both groups, BPD patients differed from HC in their neural processing of physical pain depending on the preceding social situation. Rejection sensitivity further modulated the impact of social exclusion on neural pain processing in BPD, but not in healthy controls.