Description and characterization of a chamber for viewing and quantifying cancer cell chemotaxis

Direct observations of cancer cell invasion underscore the importance of chemotaxis in invasion and metastasis. Yet, there is to date, no established method for real-time imaging of cancer chemotaxis towards factors clinically correlated with metastasis. A chamber has been designed and tested, called the Soon chamber, which allows the direct observation and quantification of cancer cell chemotaxis. The premise for the design of the Soon chamber is the incorporation of a dam, which creates a steep gradient while retaining stability associated with a pressure-driven system. The design is based on the characteristics of cancer cell motility such as relatively low speeds, and slower motility responses to stimuli compared to classical amoeboid cells like neutrophils and Dictyostelium. We tested MTLn3 breast carcinoma cells in the Soon chamber in the presence of an EGF gradient, obtaining hour-long time-lapses of chemotaxis. MTLn3 cells migrated further, more linearly, and at greater speeds within an EGF gradient compared to buffer controls. Computation of the degree of orientation towards the EGF/buffer source showed that MTLn3 cells were significantly more directional toward the EGF gradient compared to buffer controls. Analysis of the time-lapse data obtained during chemotaxis demonstrated that two populations of cancer cells were present. One population exhibited oscillations in directionality occurring at average intervals of 12 min while the second population exhibited sustained high levels of directionality toward the source of EGF. This result suggests that polarized cancer cells can avoid the need for oscillatory path corrections during chemotaxis.     

Effect of chlorsulfuron on ethylene evolution from sunflower seedlings

The effect of the herbicide chlorsulfuron (2-chloro-N-[(4-methoxy - 6 - methyl -1, 3,5 - triazin - 2 - yl)aminocarbonyl]benzenesulfonamide) on ethylene production in light-grown sunflower (Helianthus annuus L.) seedlings was examined. Application of chlorsulfuron to the apex stimulated ethylene production in all tissues examined: cotyledons, hypocotyls, and roots. The greatest stimulation occurred in the upper portion of the hypocotyl adjacent to, and including, the cotyledonary node. Ethylene evolution from hypocotyls excised from treated seedlings was stimulated over control levels 1 day after herbicide application and reached a maximum (approx. 75 x control or 17 nl/g f wt/h) 2 to 3 days after treatment. Labeling and inhibitor studies indicated that the ethylene produced was derived primarily from methionine. Chlorsulfuron treatment stimulated the rate of accumulation of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), as well as the ability of the tissue to convert exogenous ACC to ethylene. Chlorsulfuron had little effect on ethylene production when administered to the hypocotylsin vitro. Removal of the cotyledons from treated seedlings reduced the rate of ethylene evolution from the hypocotyls. These results suggest that stimulation of ethylene production in sunflower hypocotyls by chlorsulfuron is not a wound response but rather is dependent on factors derived from the cotyledons.     

ARID3B Directly Regulates Ovarian Cancer Promoting Genes

The DNA-binding protein AT-Rich Interactive Domain 3B (ARID3B) is elevated in ovarian cancer and increases tumor growth in a xenograft model of ovarian cancer. However, relatively little is known about ARID3B''s function. In this study we perform the first genome wide screen for ARID3B direct target genes and ARID3B regulated pathways. We identified and confirmed numerous ARID3B target genes by chromatin immunoprecipitation (ChIP) followed by microarray and quantitative RT-PCR. Using motif-finding algorithms, we characterized a binding site for ARID3B, which is similar to the previously known site for the ARID3B paralogue ARID3A. Functionality of this predicted site was demonstrated by ChIP analysis. We next demonstrated that ARID3B induces expression of its targets in ovarian cancer cell lines. We validated that ARID3B binds to an epidermal growth factor receptor (EGFR) enhancer and increases mRNA expression. ARID3B also binds to the promoter of Wnt5A and its receptor FZD5. FZD5 is highly expressed in ovarian cancer cell lines, and is upregulated by exogenous ARID3B. Both ARID3B and FZD5 expression increase adhesion to extracellular matrix (ECM) components including collagen IV, fibronectin and vitronectin. ARID3B-increased adhesion to collagens II and IV require FZD5. This study directly demonstrates that ARID3B binds target genes in a sequence-specific manner, resulting in increased gene expression. Furthermore, our data indicate that ARID3B regulation of direct target genes in the Wnt pathway promotes adhesion of ovarian cancer cells.     

Beta8 integrin binds Rho GDP dissociation inhibitor-1 and activates Rac1 to inhibit mesangial cell myofibroblast differentiation

Alpha(v)beta8 integrin expression is restricted primarily to kidney, brain, and placenta. Targeted alpha(v) or beta8 deletion is embryonic lethal due to defective placenta and brain angiogenesis, precluding investigation of kidney alpha(v)beta8 function. We find that kidney beta8 is localized to glomerular mesangial cells, and expression is decreased in mouse models of glomerulosclerosis, suggesting that beta8 regulates normal mesangial cell differentiation. To interrogate beta8 signaling pathways, yeast two-hybrid and co-precipitation studies demonstrated beta8 interaction with Rho guanine nucleotide dissociation inhibitor-1 (GDI). Selective beta8 stimulation enhanced beta8-GDI interaction as well as Rac1 (but not RhoA) activation and lamellipodia formation. Mesangial cells from itgb8-/- mice backcrossed to a genetic background that permitted survival, or gdi-/- mice, which develop glomerulosclerosis, demonstrated RhoA (but not Rac1) activity and alpha-smooth muscle actin assembly, which characterizes mesangial cell myofibroblast transformation in renal disease. To determine whether Rac1 directly modulates RhoA-associated myofibroblast differentiation, mesangial cells were transduced with inhibitory Rac peptide fused to human immunodeficiency virus-Tat, resulting in enhanced alpha-smooth muscle actin organization. We conclude that the beta8 cytosolic tail in mesangial cells organizes a signaling complex that culminates in Rac1 activation to mediate wild-type differentiation, whereas decreased beta8 activation shifts mesangial cells toward a RhoA-dependent myofibroblast phenotype.     

A macronutrient optimization platform for micropropagation and acclimatization: using turmeric (<Emphasis Type=Italic>Curcuma longa</Emphasis> L.) as a model plant

Tissue culture medium is often overlooked as a factor in plant biotechnology. Most work uses Murashige and Skoog (MS; Physiol Plant in 15:473–497, 1962) inorganic medium formulation, which is not likely optimal for many of the plant systems where it is used. This current study of macronutrient factors simultaneously altered media volume and amount of tissue (plants per vessel), sucrose, nitrogen (as NO₃⁻ and NH₄⁺ ions), and K⁺ in a d-optimal design space with only 55 experimental units (including five true replicates). Meso- and micro-nutrient concentrations were lowered (5% of MS) to determine which elements were most critical to plantlet quality. Plantlet quality was quantified by multiplication in the laboratory and survival and growth in the greenhouse. Plantlets grown at the lowest plant density, the lowest macronutrient concentration (20 mM), and equi-molar proportions of NH₄⁺/K⁺ resulted in the best multiplication ratio and 100% greenhouse survival. Multiplication ratio in vitro and survival in the greenhouse were well correlated with one another. Laboratory dry mass, media use, sucrose use, and the uptake of the macronutrients NO₃⁻, NH₄⁺, and K⁺ were not well correlated with plantlet quality. Plantlets with the greatest uptake of P, Ca, Mg, and Mn had the best multiplication in the laboratory and on subsequent transfer, acclimatized and grew fastest in the greenhouse. Phosphorus was shown to be most depleted in media. This work demonstrates a platform to simultaneously optimize several nutritive components of tissue culture media to produce plantlets that perform well in both laboratory and greenhouse environments. Plant quality was related with factors outside the macronutrient design, and this platform indicated where to expand the experimental space. Fixed, flat-screen presentations revealed less of the response surface than interactive profiles driven by the reader.     

A pharmacogenomic method for individualized prediction of drug sensitivity

Identifying the best drug for each cancer patient requires an efficient individualized strategy. We present MATCH (M erging genomic and pharmacologic A nalyses for T herapy CH oice), an approach using public genomic resources and drug testing of fresh tumor samples to link drugs to patients. Valproic acid (VPA) is highlighted as a proof‐of‐principle. In order to predict specific tumor types with high probability of drug sensitivity, we create drug response signatures using publically available gene expression data and assess sensitivity in a data set of >40 cancer types. Next, we evaluate drug sensitivity in matched tumor and normal tissue and exclude cancer types that are no more sensitive than normal tissue. From these analyses, breast tumors are predicted to be sensitive to VPA. A meta‐analysis across breast cancer data sets shows that aggressive subtypes are most likely to be sensitive to VPA, but all subtypes have sensitive tumors. MATCH predictions correlate significantly with growth inhibition in cancer cell lines and three‐dimensional cultures of fresh tumor samples. MATCH accurately predicts reduction in tumor growth rate following VPA treatment in patient tumor xenografts. MATCH uses genomic analysis with in vitro testing of patient tumors to select optimal drug regimens before clinical trial initiation.     

Properties of the reaction center of the thermophilic purple photosynthetic bacterium Chromatium tepidum

Reaction centers were purified from the thermophilic purple sulfur photosynthetic bacterium Chromatium tepidum. The reaction center consists of four polypeptides L, M, H and C, whose apparent molecular masses were determined to be 25, 30, 34 and 44 kDa, respectively, by polyacrylamide gel electrophoresis. The heaviest peptide corresponds to tightly bound cytochrome. The tightly bound cytochrome c contains two types of heme, high-potential c-556 and low-potential c-553. The low-potential heme is able to be photooxidized at 77 K. The reaction center exhibits laser-flash-induced absorption changes and circular dichroism spectra similar to those observed in other purple photosynthetic bacteria. Whole cells contain both ubiquinone and menaquinone. Reaction centers contain only a single active quinone; chemical analysis showed this to be menaquinone. Reaction center complexes without the tightly bound cytochrome were also prepared. The near-infrared pigment absorption bands are red-shifted in reaction centers with cytochrome compared to those without cytochrome.