Nidation of floating tumour cells initiates peritoneal carcinosis and limits prognosis of gastro-intestinal tumours. Adhesion of tumour cells to extracellular matrix components is a pivotal step in developing peritoneal dissemination of intraabdominal malignancies. Since phospholipids efficaciously prevented peritoneal adhesion formation in numerous animal studies we investigated their capacity to reduce adhesions of gastric cancer cells to extracellular matrix components (ECM).Human gastric cancer cells  were used in this study. Microtiter plates were coated with collagen IV (coll), laminin (ln) and fibronectin (fn). Non-specific protein binding of the coated wells was blocked by adding 1% (w/v) BSA  and rinsing the wells with Hepes buffer. 50.000 tumour cells in 100 μl medium were seeded into each well. Beside the controls, phospholipids were added in concentrations of 0.05, 0.1, 0.5, 0.75 and 1.0/100 μl medium. After an incubation interval of 30 min, attached cells were fixed and stained with 0.1% (w/v) crystal violet. The dye was resuspended with 50 μl of 0.2% (v/v) Triton X-100 per well and colour yields were then measured by an ELISA reader at 590 nm. Optical density (OD) showed a linear relationship to the amount of cells and was corrected for dying of BSA/polystyrene without cells.The attachment of gastric cancer cells to collagen IV, laminin, and fibronectin could be significantly reduced up to 53% by phospholipid concentrations of 0.5 mg/100 μl and higher.These results, within the scope of additional experimental studies on mice and rats which showed a significant reduction of peritoneal carcinosis, demonstrated the capacity of phospholipids in controlling abdominal nidation of tumour cells to ECM components. Lipid emulsions may be a beneficial adjunct in surgery of gastrointestinal malignancies. In the treatment of gastro-intestinal cancer the detection of free, isolated tumour cells in the peritoneal cavity serve as a prognostic marker for postoperative survival -4. Sincein vitro study was focused on the influence of phospholipids on adhesion of gastric cancer cells to extracellular matrix components with broad reactivity to several integrins. Collagen IV (coll IV), and laminin (ln) are main components of the basement membrane and fibronectin (fn) plays an important role in wound healing [Phospholipids, polar phosphoric acid di-esters, are natural constituents of the abdominal fluid. The substance is able to form a lubricant layer on the peritoneal surface . Additio healing ,18.2, Falcon, Becton Dickinson-Gambil, Heidelberg, Germany) in RPMI 1640 medium , supplemented with 10% foetal bovine serum (GIBCO), penicillin and streptomycin (GIBCO). Cell cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air. Cells were passaged after treatment with 0.125% trypsin for 6 min. The cells were pelleted after centrifugation for 10 min at 200 g, suspended in 20 ml PBS, and pelleted. The cell pellet was resuspended in 30 ml complete medium and seeded with a splitting ratio of 1:3. Only cells from three passages were used for the experiments.The human gastric cancer cell line NUGC-4 was purchased from the Japanese Cancer Research Resources Bank . The cells were maintained in monolayers in tissue culture flasks (75 cm2 in air (ln), respectively. Nonspecific protein binding of the coated wells was blocked by adding 1% (w/v) BSA  and rinsing the wells with Hepes buffer.Flat-bottom polystyrene microtiter plates  were coated for adhesion experiments. The purified ECM components were dissolved in PBS with the following concentrations: coll IV – 2,5 μg/ml , fn – 10 μg/ml , ln – 50 μg/ml . We found these concentrations to be optimal in foregoing dilution series. They were added to the wells and incubated at 4°C for 24 hours , or at 37°C for 45 min in a humidified atmosphere of 5% CO3 and 5 × 104 cells per well, as determined by a dilution series.For adhesion experiments gastric cancer cells were detached with collagenase I , washed once with RPMI 1640, centrifuged (200 g for 10 min), resuspended in RPMI 1640, and preincubated for 30 min in a humidified atmosphere of 5% CO2 in air (37°C). Fifty thousand tumour cells in 100 μl medium were seeded into each well. Evaluation of adherent cells was performed using crystal violet staining according to the method described by Aumeilley et al., and Tietze et al. ,20. AfteControl dying of BSA/polystyrene without cells led to Optical Density (OD) values of 0.01–0.07. These values were subtracted from those obtained in the experiments.After complete preparation of the tumour cell suspension, the PL solution was added in the following concentrations: 0.05, 0.1, 0.5, 0.75, and 1 mg per 100 μl medium. The concentrations used were correlated to our in vivo experiments. The phospholipid solution consists of phosphatidylcholine 70% by weight, phosphatidylethanolamine 15% by weight, neutral lipids 8% by weight, sphingomyelin <3% by weight and lysophosphatidylcholine <3% by weight.All experiments were performed three times in quadruplicate. The data are expressed as means +/- standard error of the mean (SEM). Student's t-test for unpaired data was used for statistical analysis. Differences were regarded as significant for p values < 0.05.The analysis of tumour cell adhesion to BSA 1% resulted in a mean extinction of 0.27 (SEM 0.01) at 590 nm. Coating with ln and fn led to a nearly twofold increase of tumour cell adhesion with mean values of 0.59  and 0.63 . The cancer cells showed a most pronounced adhesion to coll IV with a mean extinction of 0.97 (0.02).The tumour cell adhesion to ln registered after addition of PL was significantly reduced. The effect was concentration dependent compared to the controls. Even the minimum amount of PL 0.05 mg/100 μl led to a reduced extinction of 0.4 (0.01). Treatment with 0.1 or 0.5 mg/100 μl PL revealed extinction values of 0.32 (0.02) and 0.28 (0.02), respectively. The maximum effect could be demonstrated with 0.75 mg/100 μl PL with an extinction of 0.24 (0.02). The relative reduction of tumour cell adhesion compared to the control amounts to 59%. Treatment with 1 mg/100 μl PL showed no further decrease of tumour cell adhesion to ln. The mean extinction was 0.26 (0.01)  and 0.59 (0.01). However, a significant reduction of tumour cell adhesion could be observed after treatment with 0.5 mg/100 μl PL, 0.42 (0.02); as well as with 0.75 mg/100 μl PL (0.39 (0.02)) and 1 mg/100 μl PL (0.38 (0.02)). We found a similar situation compared to ln with equal effects of 0.75 mg/100 μl and 1 mg/100 μl PL indicating that the maximum influence on adhesion is reached. The relative reduction of tumour cell adhesion compared to the control values amounts to 40%  after administration of 0.05 mg/100 μl PL to a maximum effect after treatment with 1 mg/100 μl PL with a value of 0.44 (0.02) table . In compCell adhesion to the extracellular matrix plays a fundamental role in peritoneal carcinosis. The adhesion is mediated by transmembrane Integrins. Several proteins including fibronectin in the interstitial matrix, laminin and collagen IV in the basement membrane were identified as important ligands ,22. Manyin vitro as well as a reduction of tumour weight after intraperitoneal tumour injection [In our experiments the reduced rate of cell attachment in the presence of phospholipids was independent from the extracellular matrix. A similar effect on intraperitoneal tumour growth was described by Jacobi et al. who could demonstrate that taurolidine/heparin and povidone iodine lead to a significant reduction of tumour cell growth njection . Predominjection . Dextrannjection ,32. Howenjection ,34. PhosThe hypothesis is that phospholipids form a lubricant layer on the peritoneum by binding with its negatively charged cholin branch chain to the positively charged peritoneal surface ,14,35. Pin vitro experiments with tumour cells as soluble agents added to ECM immobilized onto plastic surfaces cannot appropriately mimic the situation in situ. Recently we found that phospholipids significantly reduce the attachment area and the tumour volume of peritoneal carcinosis caused by the colonic cancer cell line DHD/K12/TRb in rats. These results were supported by a prolonged survival rate of the treated animals as compared to the control group. Additionally, we found a similar effect of phospholipids on the adhesion of the human rectal cancer cell line HRT-18 on the same ECM-components in vitro [The in situ tumour cell – ECM interaction is influenced by adhesive and non-adhesive ECM components and can be understood as a three dimensional network . Therefoin vitro . Consistin vitro ,6,38,39.We performed this study to ascertain the results of the foregoing animal experiments and to demonstrate the influence of phospholipids to three different ECM components, even though matrices of collagen IV, laminin and fibronectin alone may not be predictive of peritoneal membrane nidation.These results, within the scope of additional experimental studies on mice and rats which showed a significant reduction of peritoneal carcinosis, demonstrated the capacity of phospholipids in controlling abdominal nidation of tumor cells to ECM components. Lipid emulsions may be a beneficial adjunct in surgery of gastrointestinal malignancies.The work was financially supported by Fresenius Kabi, Bad Homburg, Germany.The results are part of an international patent application.The pre-publication history for this paper can be accessed here:


To function properly, cells must keep constant tabs on the environmental conditions around them, such as the presence of growth hormones in the blood or the proximity of neighboring cells. These external cues are relayed into the cell through a cascade of chemical and physical reactions referred to as signal transduction. Signal transduction pathways inform and regulate almost all activity within the cell, from protein production to cell division. Understanding these processes is fundamental to biology, but the sheer number of molecules and interactions in some pathways makes thorough documentation difficult.Taking a holistic approach that combines both computational models and experimental manipulations, scientists have described the web of interactions involved in the aryl hydrocarbon receptor (AHR) signal transduction pathway. AHR belongs to the Per–Arnt–Sim (PAS) superfamily of sensor molecules that regulate functions like development, the sleep-wake cycle, and cellular reaction to oxygen deprivation. Unlike many receptors that are embedded in the cell membrane, AHR floats freely in the main body of the cell, called the cytosol. There it waits for a stimulus or ligand, such as a dioxin molecule, to enter the cell and bind to it. Once bound, AHR undergoes a host of changes, glomming on to additional molecules before it enters the cell nucleus and acts as a transcription factor, initiating the production of enzymes to digest foreign, or xenobiotic, compounds. The AHR pathway is a curiosity; though found in all vertebrates, the natural, or endogenous, ligand remains unknown. Without this knowledge, researchers are limited in the kind of experiments they can perform to evaluate the pathway.Christopher Bradfield and colleagues used yeast as a model system to elucidate the steps involved in this pathway, which regulates vertebrate cell response to pollutants like dioxins. To first assess the molecules involved in the AHR pathway, the team used 4,507 yeast “deletion” strains, each strain missing one gene from its genome. They then inserted the AHR gene into the strains using small rings of movable DNA called plasmids. Though yeast does not naturally possess AHR, it is an ideal genetic model for studying signaling pathways due to its quick generation time, small, well-characterized genome, and similarity to vertebrate systems.Bradfield's team exposed each strain to a receptor stimulus or agonist and screened them for AHR response. If a deletion strain showed significantly reduced activity, they concluded that the missing gene was a key component to the signal pathway. The researchers identified 54 genes that had a significant influence on AHR response. Only two of these genes, termed modifiers, had been previously identified.Signaling pathways usually boil down to a series of discrete steps. To identify steps of the AHR pathway, the researchers constructed a spider web-like map called a "protein interaction network," or PIN, based on previously known interactions between the proteins encoded by the 54 modifier genes. The resulting map revealed groups of highly connected, related modifiers, which the authors proposed to be steps in the pathway. Though other studies have used the newly developed PIN strategy to investigate cellular processes, Bradfield's team also annotated their PIN through a series of experiments both to support the identity of and to better understand the protein groups, referred to as functional modules.With tests based on discrete receptor signaling events, known active structural regions, reaction to different types and concentrations of agonists, and functional location within the cell, Bradfield's team organized the functional modules into five steps. One group of modifiers is involved in AHR folding, the conformational change that occurs when the receptor binds to a toxin. With the help of other modifiers, the new AHR complex is then translocated into the cell nucleus. Once in the nucleus, a series of modifiers assist the AHR in its role as a transcription factor. The researchers also identified a step in the pathway that controls production of AHR itself and another unknown "step" that takes place inside the nucleus.As AHR is thought to be a prototype PAS receptor, understanding the steps in this pathway will likely guide future research on the entire family, allowing scientists to study in detail individual steps in these complex pathways. The highly integrated method reported here could also be used to study most other mammalian signaling pathways, giving scientists a new tool as they attempt to understand how cells respond to their changing environment.


DJ-1 were recently described in two families with autosomal recessive inherited PD (Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in rited PD . In a corited PD , we show The interaction of the proteins DJ-1 and α- synuclein described here may be important for understanding the molecular basis of Parkinson's disease Parkin, lead to autosomal recessive primary Parkinsonism (Parkinson's disease (PD) is a progressive movement disorder that is characterized pathologically by the relatively selective degeneration of midbrain DNs and the presence of prominent intracytoplasmic neuronal inclusions, termed Lewy bodies . The ideinsonism . Parkin insonism .DJ-1, were recently associated with autosomal recessive primary Parkinsonism , many of which function as molecular chaperones to assist other proteins in folding. Hsp31, an in vitro . We hypoin vitro A. The chin vitro B.DJ-1 similarly functioned as a molecular chaperone in the context of the heat-induced aggregation of GST see . In cont2O2 , and subsequently chaperone activity was measured in the CS thermal aggregation assay. H2O2 effectively reactivated the chaperone activity of DTT-treated DJ-1 . These results suggest that redox regulation of DJ-1 is reversible and is regulated by the redox environment.To test the redox regulation of DJ-1, we assayed chaperone activity in the CS aggregation assay in the presence or absence of the reducing agent DTT. DJ-1 chaperone activity in the CS aggregation assay was completely abrogated by preincubation of DJ-1 with 0.5 mM DTT in aggregation buffer for 10 min at 4 °C D. DTT dited DJ-1 D. This wMolecular chaperones typically display marked stability to thermal stress . ConsistWe extended the analysis of DJ-1 chaperone function to a candidate DJ-1 substrate, αSyn . The aggαSyn protofibrils have been shown to be an intermediate in the formation of mature amyloid fibrils. Because DJ-1 chaperone activity is effective at inhibiting the accumulation of αSyn protofibrils, we sought to investigate the role of this activity in the generation of Congo red–positive mature fibrils. Congruently, WT DJ-1 inhibited formation of Congo red-positive αSyn fibrils, while L166P DJ-1 and GST did not C. Thus, 2 treatment ) were recorded on an Aviv 62A sCD spectrometer at 4 °C in a 0.02-cm path length cuvette, and α-helix and β-sheet content were estimated as described (2 · dmol−1) at 222 nm as a function of temperature. Thermal melts were performed in 4 °C increments with an equilibration time of 1 min and an integration time of 30 sec, using a 0.1-cm path length cuvette.(E) Far-ultraviolet CD spectra of WT DJ-1 (blue triangles) and the L166P mutant (red squares); mean residue ellipticity (Θ) equals °C · cmescribed . Based o222 is equal to °C · cm2 · dmol−1 at 222 nm.(F) Thermal denaturation curves for WT and mutant L166P DJ-1; mean residue ellipticity (Θ)(G) Redox regulation is unaffected by the C106A mutation. Redox regulation of C106A DJ-1 was assayed via DTT inactivation (0.5 mM) in the CS aggregation suppression assay.(H) Protofibril preparations (as in (1.2 MB PDF).Click here for additional data file.Figure S22 (Fe) or media alone (0) as described in (A) Undifferentiated ES cells were transfected with Flag-αSyn and treated with 2 mM FeCl5 cells in a 24-well format were transiently transfected with eukaryotic expression constructs encoding WT human DJ-1 or empty vector. After 36 h, cells were starved for 1 h with DMEM lacking cysteine and methionine and supplemented with 8% dialyzed FBS. Cells were pulsed for 2 h with 10 μCi[35S]-L-Met/L-Cys  per well, washed twice, and chased at the indicated intervals with complete medium. Flag-αSyn was immunoprecipitated with Flag antibody-conjugated agarose beads (Sigma), subjected to SDS-PAGE, and visualized by autoradiography.(B) Overexpression of WT DJ-1 does not significantly alter the half-life of soluble Flag-αSyn. CAD murine neuroblastoma cells were stably transfected with Flag-tagged human α-synuclein using standard techniques. 2 × 10(C) Flag-αSyn from (B) was quantitated using NIH Image J.(815 KB PDF).Click here for additional data file.Figure S3p < 0.(A) Overexpression of WT DJ-1 or L166P DJ-1 in the context of αSyn aggregation does not alter cell number. Cells from p < 0.(B) Overexpression of WT DJ-1 or L166P mutant DJ-1 in the context of Q333P mutant NFL aggregation does not alter cell number. GFP positive transfected cells from p < 0.(C) Overexpression of WT DJ-1, but not L166P mutant DJ-1, rescues cells from Q333P mutant NFL toxicity. HeLa cells were transfected with Q333P mutant NFL along with WT human DJ-1, L166P mutant DJ-1, or vector control. After 72 h, cells were assayed by MTT reduction assay -2,5-diphenyltetrazolium bromide by metabolic enzymes) . Data arp < 0.(D) C53A mutant DJ-1 is unable to rescue cells from Q333P mutant NFL toxicity. Undifferentiated ES cells were transfected with Q333P mutant NFL along with WT human DJ-1, C53A mutant DJ-1, or vector control. After 72 h, cells were assayed by MTT reduction assay . Data ar(E) Coexpression of DJ-1 with NFL does not alter NFL expression levels. CAD cells were transfected with Q333P mutant NFL and vector, WT DJ-1, C53A mutant DJ-1, or L166P mutant DJ-1. Cells were differentiated for 72 h and lysed to produce Triton X-100-soluble and -insoluble fractions. Lysates were exposed to Western blotting with an antibody against transfected human NFL. NFL is present only in the insoluble fraction, and expression of WT or mutant DJ-1 does not alter NFL expression levels.(685 KB PDF).Click here for additional data file.Figure S42 (G–L) for 18 h prior to fixation with PFA. Cells were immunostained with rabbit anti-DJ-1 as described, followed by donkey anti-rabbit Cy5 . Nuclei  were visualized by incubation with the nuclear stain ToPro3 prior to imaging.CAD cells were transfected with WT DJ-1 and differentiated by serum withdrawal for 72 h. Cells were treated with medium alone (A–F) or medium with 2 mM FeCl(1.6 MB PDF).Click here for additional data file.Table S1(45 KB DOC).Click here for additional data file.


Several cell lines and primary cultures benefit from the use of positively charged extracellular matrix proteins or polymers that enhance their ability to attach to culture plates. Polyethyleneimine is a positively charged polymer that has gained recent attention as a transfection reagent. A less known use of this cationic polymer as an attachment factor was explored with several cell lines.Polyethyleneimine compared favorably to traditional attachment factors such as collagen and polylysine. PC-12 and HEK-293 cells plated on dishes coated with polyethyleneimine showed a homogeneous distribution of cells in the plate, demonstrating strong cell adhesion that survived washing procedures. The polymer could also be used to enhance the adherence and allow axonal outgrowth from zebrafish retinal explants. The effects of this coating agent on the transfection of loosely attaching cell lines were studied. Pre-coating with polyethyleneimine had the effect of enhancing the transfection yield in procedures using lipofection reagents.Polyethyleneimine is an effective attachment factor for weakly anchoring cell lines and primary cells. Its use in lipofection protocols makes the procedures more reliable and increases the yield of expressed products with commonly used cell lines such as PC-12 and HEK-293 cells. Molecular cell biology experimentation often requires the culture of primary cells or immortalized cell lines. The most common substratum used in cell culture consists of a plastic dish that offers a negatively charged surface. A drawback of this technology is that some anchorage-dependent cell types do not produce sufficient amounts of positively charged extracellular matrix proteins, adhering only weakly to the plastic substratum. Pre-coating of the plastic surface with extracellular matrix proteins such as collagen, fibronectin, laminin, etc., usually enhances the attachment of these cell types ,2. SynthPolyethyleneimine (PEI) is an organic polymer that has a high density of amino groups that can be protonated. At physiological pH, the polycation is very effective in binding to DNA and can mediate the transfection of eukaryotic cells . The oriThis report presents a comparison of PEI with other traditional coating agents as attachment factors for several cell lines and primary cultures of retina tissue. Additionally, an enhancement of the transfection yield of weakly anchoring cell lines is shown by the combination of PEI coating with lipofection.PC-12 cells are used as a model of neuronal differentiation in the laboratory, as treatment of these cells with nerve growth factor (NGF) induces neurite extension and the expression of biochemical markers of the sympathetic neuronal phenotype . They grin vitro by the accelerated extension of long neurites. However, this phenomenon requires the use of extracellular matrix or attachment factors (for example collagen or PDL coating), as the explants show very low affinity for the uncoated plastic surface. Experiments were conducted to observe if PEI could act as an attachment factor conducive to axonal outgrowth from zebrafish retinal explants. Retinal explants from control zebrafish eyes were able to attach to PEI-pretreated culture dishes . Plates pretreated with the diverse attachment factors were compared with plates that received no pretreatment (untreated). Experiments were performed in triplicate, and the averages of dye retained in the plates that received each treatment were calculated. Those averages were normalized to the average obtained with the PEI pretreatment, which was assigned the arbitrary value of 100.0% in all the experiments. The results from representative experiments with the 3 cell lines are shown in Figure To provide an indication of the variation among experiments, the averages ± standard deviations of the relative cell counts obtained in the independent experiments were calculated for each cell line and treatment . The comparative results for the other treatments are indicated below. For the PC-12 cells, the relative counts were 81.5% ± 8.8% for collagen-pretreated wells (n = 4), 93.9% ± 21.2% for PDL-pretreated wells (n = 4), and 52.1% ± 13.7% for untreated wells (n = 4). For the HEK-293 cells, the relative counts were 16.3% ± 12.7% for collagen-pretreated wells (n = 3), 99.6% ± 2.5% for PDL-pretreated wells (n = 3), and 9.0% ± 4.1% for untreated wells (n = 3). In the experiments with MYS cells, the average of the relative counts with collagen-pretreated wells was 92.7% ± 16.2% (n = 3), 71.1% ± 6.7% for PDL-pretreated wells (n = 3), and 78.4% ± 11.5% for untreated wells (n = 3). Statistical analysis indicates that the enhancement of adhesion of both PC-12 and HEK-293 cells to PEI-pretreated dishes versus untreated plastic is significant , while it is not statistically significant for MYS cells (p > 0.05).5 HEK-293 cells and 1.5 × 106 PC-12 cells). The results indicate that PEI worked well as an attachment factor over a wide range of cell numbers for both PC-12 and HEK-293 cells. Lower cell numbers could not be tested reliably because they were close to the limit of sensitivity of the neutral red assay. The results shown are representative of at least 4 independent experiments performed with each cell line. Figure 2 incubator for 3 days, with a medium change performed every 24 h. The performance of PEI in these plates was then compared with plates treated with PEI using the standard protocol described for previous experiments. The results show relative cell counts normalized to the average of the absorbances obtained with the standard PEI-treated plates, which was given the arbitrary value of 100.0%. The results indicate that the PEI coating remains stable on the surface of the plastic dish for at least 10 days of refrigeration, and that it is not removed by incubation at 37°C in medium or even by repeated medium changes. Results are representative of 4 independent experiments performed in triplicate.To further characterize the properties of PEI as an attachment factor for weakly anchoring cells, experiments were carried out to analyze the dosage of PEI that can provide optimal cell anchoring, the range of cell numbers that can benefit from the presence of PEI, and the stability of PEI as attachment factor. Figure Transfection of eukaryotic cells by lipofection involves several steps, media additions and replacements, which can take a toll in weakly anchoring cells. Even if care is practiced to avoid cell loses, the weakly anchored cells may be less efficient in the uptake of the transfection complexes. Since PEI pretreatment of cell culture dishes increased the strength of attachment of cells to such surfaces, it was hypothesized that the attachment factor may have a positive effect in transfection yields obtained by lipofection. The three cell lines described above were used to test this hypothesis utilizing the coating agents previously examined. Transfections were performed with a plasmid encoding the reporter enzyme β-galactosidase. The transfection yield was monitored by determination of reporter enzyme activity in lysates from the transfected cells. At least two independent assays in triplicate were performed with each cell line. Representative plots are shown in Figure Compiling the results from all the experiments performed, the average fold-induction (± standard deviation) observed in the transfection yield of PC-12 cells anchored to PEI as compared with cells on untreated wells was 2.4-fold , while the enhancement with HEK-293 cells was 6.3-fold . t-test statistical analysis indicates that both increases are significant (p < 0.05). No significant transfection enhancement was observed in the experiments with MYS cells, with an average fold-change of 1.0-fold  by the pretreatment with PEI .4 to 3.2 × 105 cells per well. When larger numbers of HEK-293 cells were utilized the results were more variable, suggesting that when these cells are near confluency they may be able to form monolayers that attach more firmly to the plate, however further experiments are necessary to study this hypothesis. The effects of PEI and the other cationic attachment factors on PC-12 and HEK-293 cells suggest that these cells have a limited capacity to produce an effective extracellular matrix to allow them to attach to the plastic surface, resulting in weak anchoring to the dishes on their own. Cationic polymers such as PEI and PDL seem to work very effectively to coat the surface of the dish and substitute for the absence of the extracellular matrix components. On the other hand, fibroblast cells (such as the MYS cells) are very efficient producing extracellular matrix proteins, which allows them to attach firmly to the culture dishes, therefore no benefit was observed by the use of attachment factors with these cells , but this is not so for protocols with HEK-293 cells. PEI was very effective in improving lipofection protocols with both cell lines. The firm attachment afforded by PEI allowed transfections with cationic lipids (lipofection) to provide higher yields and more consistent results.2 atmosphere in a tissue culture incubator (NuAire). Zebrafish retinal explants were maintained in L-15 complete medium: L15 medium (Sigma) supplemented with 1% fetal bovine serum (Hyclone), 100 u/ml penicillin, 100 μg/ml of streptomycin and 2 mM glutamax . The explants were incubated at 28°C at normal atmospheric CO2 concentrations.PC-12  cells were cultured with RPMI complete medium: RPMI medium (Invitrogen) supplemented with 10% horse serum and 5% fetal bovine serum (both from Hyclone), 100 u/ml of penicillin, 100 μg/ml of streptomycin, 10 mM HEPES, 2 mM glutamax and 1 mM sodium pyruvate . HEK-293  and MYS  cells were cultured in DMEM complete medium: DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (Hyclone), 100 u/ml of penicillin, 100 μg/ml of streptomycin and 2 mM glutamax . Cell lines were maintained at 37°C in a 5% CO6 cells) was added to each well of the MW12 and the plate was incubated at 37°C in a 5% CO2 atmosphere for 48 h. The cells were then examined under a phase contrast Olympus-CK40 microscope and photomicrographs were obtained with a Pixera Penguin 150 CL camera. The experiment was performed in triplicate and pictures were taken at the center, near the edge and midway between center and edge of the plate, to evaluate the distribution of cells on the wells.For the culture and microscopic observation of PC-12 cells, the wells of a multiwell-12 tissue culture dish  were pretreated for 20 min with 100–200 μl of the different solutions of attachment factors: PEI-800 kDa, from Fluka, at 25 μg/ml in 150 mM NaCl; PDL, from ICN, at 100 μg/ml in deionized water; bovine dermal collagen, from ICN, 3 mg/ml aqueous solution. Usually, each column of wells in the MW12 was pretreated with one of the attachment factors, while the last column was left untreated. After removal of the different attachment factor solutions, 1 ml of a PC-12 cell suspension  was performed retro-orbitally with anesthetized zebrafish 10 days prior to explantation, using protocols previously described ,25. Cont5 PC-12 cells and cultured with RPMI complete medium as previously described. After 24 h, the medium was replaced by RPMI medium with low serum , which was supplemented with NGF (Sigma) at 100 ng/ml to induce differentiation. The culture medium was replaced by new RPMI low serum medium with 100 ng/ml of NGF every two days. At different times during the differentiation process , photomicrographs were obtained as previously described.The wells of a MW12 were pretreated with PEI as described above. The wells were then seeded with 2.5 × 106 cells per well for the experiments with PC-12 cells, 1.5 × 105 cells for the experiments with HEK-293 cells, or 3 × 104 cells for the experiments with MYS cells. The MW12 dishes were incubated at 37°C in a 5% CO2 atmosphere for 48 h in the case of PC-12 cells, or 24 h for HEK-293 and MYS cells. In all the experiments, the wells were washed 4 times with phosphate buffered saline (PBS) to test the strength of anchorage to the substratum. To estimate the number of cells remaining after the multiple washings, a procedure that utilizes the vital dye neutral red was employed [Pretreatment of MW12 dishes with the attachment factors was performed as described above. PC-12, HEK-293 and MYS cells were counted using a Neubauer hemocytometer, and the MW12 plates were seeded with 1 × 10employed . BrieflyTwo-sample (unpaired) t-test analyses were performed with a Microsoft Excel worksheet designed for this purpose. The worksheet calculates the t-factor by comparing 2 independent sets of data and estimates the probability (p) of obtaining that result assuming that the two samples came from the same population  based on the Student's t-distribution (two-tailed). The averages of the two samples from the same experiment were considered statistically different whenever p was lower than 0.01. For comparisons of the independent experiments, one-sample t-test analyses were utilized to estimate the p values and assess the statistical significance of the difference observed between the PEI-treated wells and the untreated controls. The effect of PEI was considered significant whenever p was lower than 0.05.5 HEK-293 cells were seeded onto the dishes and incubated at 37°C for 24 h. Untreated wells were seeded in the same manner as controls.For the determination of optimal concentrations of PEI for the pretreatment procedure, a series of solutions of PEI were prepared so that their final concentrations of PEI polymer (w/v) were 0.025, 0.25, 2.5, 25 and 250 μg/ml. They were used to coat MW12 tissue culture dishes as previously described for the 25 μg/ml solution. To test the efficacy of coating of the solutions, 2 × 10The dishes were then subjected to the multiple washing procedure described above and the number of cells remaining in the dishes was measured by the neutral red dye procedure as described previously. All experiments were performed in triplicate and the relative cell counts were calculated by normalization to the values obtained with 25 μg/ml of PEI, which was assigned a value of 100.0%.4, 8 × 104, 1.6 × 105 and 3.2 × 105) or PC-12 cells . Control untreated MW12 culture dishes were seeded with the same numbers of cells in parallel with the treated plates. After 24 h for the HEK-293 cells or 48 h for the PC-12 cells, the strength of anchoring of the cells to the dishes was measured as described before and relative cell numbers were determined . All experiments were performed in triplicate.The wells of MW12 culture dishes were pretreated with 25 μg/ml of PEI as described above. The wells were then seeded with various numbers of either HEK-293 cells . The strength of attachment of cells to these dishes was determined as mentioned before, and relative cell counts were calculated by normalization to the value obtained with the standard PEI-treated wells, which was assigned arbitrarily a value of 100.0%. All experiments were performed in triplicate.To analyze the stability of the PEI attachment factor onto the plastic surface of the tissue culture dish, two tests were performed. One MW12 dish was coated with 25 μg/ml of PEI as previously described, then the PEI solution was replaced by PBS and the dish was kept at 4°C for 10 days. A second MW12 dish was coated with 25 μg/ml of PEI, then the PEI solution was replaced by DMEM complete medium and the dish was kept at 37°C in the CO2 atmosphere for 48 h for the PC-12 cells, or 24 h for the HEK-293 and MYS cells, the transfection yield was determined by measuring β-galactosidase activity in cell extracts [2HPO4, 36 mM NaH2PO4, 2 mM MgCl2 and 100 mM β-mercaptoethanol). The reaction mixture was incubated at 37°C until development of yellow coloration was apparent . Reactions were stopped by the addition of 500 μl of 1 M Na2CO3. The relative levels of β-galactosidase activity were obtained by determination of the absorbance at 420 nm in a spectrophotometer (Beckman DU500). Parallel assays with cells transfected with pcDNA3 plasmid (Invitrogen) were conducted as negative controls in every experiment, and subtracted from the experimental absorbances to correct for endogenous activities. The experiments were conducted in triplicate. Absorbances were normalized to the values obtained with PEI-pretreated wells.MW12 culture dishes were pretreated and seeded with cells as indicated above for the cell number measurements. The cells were subjected to lipofection with 1 μg per well of the plasmid pcDNA3-βgal  and Lipoextracts . The celThe authors declare that they have no competing interests.A.R.V. performed the experiments comparing the various attachment factors. S.G. and K.L.V.P. performed the PEI dosage, cell number and PEI stability analysis experiments, as well as the NGF differentiation experiments with PC-12 cells. M.J. participated in the experiments with MYS cells. R.P.B. and M.G.G. collaborated as Principal Investigators in this project, participating in the design of the experiments and the writing of the manuscript. R.P.B. performed the experiments with zebrafish retinal explants.


S. cerevisiae as a model system. Through the use of arrayed yeast strains harboring ordered deletions of open reading frames, we determined that 54 out of the 4,507 yeast genes examined significantly influence AHR signal transduction. In an effort to describe the relationship between these modifying genes, we constructed a network map based upon their known protein and genetic interactions. Monte Carlo simulations demonstrated that this network represented a description of AHR signaling that was distinct from those generated by random chance. The network map was then explored with a number of computational and experimental annotations. These analyses revealed that the AHR signaling pathway is defined by at least five distinct signaling steps that are regulated by functional modules of interacting modifiers. These modules can be described as mediating receptor folding, nuclear translocation, transcriptional activation, receptor level, and a previously undescribed nuclear step related to the receptor's Per–Arnt–Sim domain.The aryl hydrocarbon receptor (AHR) is a vertebrate protein that mediates the toxic and adaptive responses to dioxins and related environmental pollutants. In an effort to better understand the details of this signal transduction pathway, we employed the yeast  4500 yeast deletion mutants, transformed with a reporter construct that is sensitive to dioxin, were used to create a protein-interactive map that outlines five distinct steps required for dioxin-mediated cell signaling The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor found in a variety of vertebrate species. The AHR is a prototype member of the Per–Arnt–Sim (PAS) superfamily of signaling molecules. Members of this superfamily regulate cellular responses to a variety of environmental stimuli, including pollutants, hypoxia, and external light cues . Our iniCYP1A1, CYP1A2, and CYP1B1  strategy to provide a framework to describe AHR signaling. By coupling both computational and experimental annotations, we were able to deduce the minimum number of genetic loci and signaling events required for AHR signaling.Saccharomyces cerevisiae is a valuable model system for the study of signaling by mammalian nuclear receptors  and large-scale databases describing protein and genetic interactions  . This chSaccharomyces Genome Deletion Project (wt) BY4742 strain (p < 10–6). To minimize false positives, we selected clones that influenced signaling at no less than two of the six concentrations of agonist tested. In addition, we retested each positive strain in a secondary screen with another AHR system containing the same LacZ reporter and a high-copy AHR–LexA chimera (pAHR)  . By thest strain .To eliminate those deletions that influenced the AHR pathway in a nonspecific manner, each of the 92 deletion strains was examined with a control plasmid pGal4TAD see . This coOf these “AHR-specific” factors, Hsc82p and Cpr7p were previously described AHR modifiers, and the other 52 were novel  Table S. The anaRecent experiments from a number of laboratories have provided data to support the idea that protein interaction network (PIN) can be used to portray the workings of complex biological systems . To invemax). One common feature of AHR–PINs with Dmax values greater than 1 was that the majority of M-nodes were interconnected in a single large network with no breaks (max was set at low stringency (Dmax ≥ 3), the representation of M-nodes in AHR–PIN was high. For example, at Dmax = 3, 46 of 54 M-nodes were included. However, AHR–PINs resulting from these inclusive, yet low-stringency conditions exhibited high complexity, which made it impossible to assess the interactions visually (max was set at higher stringency (Dmax = 2), the resultant AHR–PIN now comprised 34 closely interconnected M-nodes and was much easier to visualize  are interconnected, while in corresponding random PINs with mock M-nodes, this number drops to 18.5% (10/54). Although the AHR–PINs at Dmax = 3 and Dmax = 4 also exhibited statistically significant differences from random PINs, these AHR–PINs were not considered further for two reasons. First, these networks were visually complex and could not be simply annotated in two dimensions. Second, the ratios of interconnected M-nodes in these AHR–PINs to those of random PINs were quite low . This observation suggests a much greater potential for displaying false positive interactions at these settings as compared to the AHR–PIN at Dmax = 2, where this ratio was 3.4 (34/10).For further exploration, we chose to focus on the network with the greatest statistical significance, i.e., the PIN generated at DOur next objective was to use the PIN to enumerate and define steps in AHR signaling. It has been suggested that PINs exhibit a modular nature, with each module comprising highly interconnected proteins of related cellular functions . Our hypmax = 2). Each distance (d) in the matrix refers to the length of the shortest path between a pair of nodes in the full network space of yeast genomic PIN and was transformed into an “association” value (21/d). The resultant pairwise association matrix was used to identify network clusters in the AHR–PIN by a hierarchical average-linkage clustering algorithm  at various agonist concentrations, times, and temperatures, as well as after exposure to two distinct AHR agonists, α-naphthoflavone (αNF) and β-naphthoflavone (βNF). The relationship between each modifier and signaling was then examined using a hierarchical average-linkage clustering algorithm  Figure A. It waswt strain was transformed with the plasmid pAHRGFP, it was found that the fusion protein was evenly distributed in the cell in the absence of AHR agonist. In the presence of the agonist βNF, the AHR–GFP protein translocated to the nucleus (wt strain (group I). Approximately 30% of the strains were found to contain a marked reduction in the level of AHR protein in the cell (group II). Approximately 10% of the deletion strains displayed receptor aggregates in the cell (group III). The final 10% of the deletion strains displayed a normal level of AHR protein, but the receptor failed to translocate into the nucleus in the presence of agonist (group IV). When overlaid with the previously determined experimental layers, group I was found to overlap with the modules of C and D, and groups II, III, and IV corresponded to modules B, A, and E, respectively . When the  nucleus A. To exa nucleus B. About ectively C. Accordectively C.Our initial objective was to identify the number of loci that are required for AHR signal transduction. In this regard, our high-throughput deletion screen identified 52 novel and two known AHR modifiers. Although this is a surprisingly large number of modifiers for the function of a single protein, it is probably an underestimate since the deletion screen cannot identify modifiers that are encoded by essential genes. Moreover, our criteria of including only strong modifiers (influence of 4-fold compared to control) may have caused us to miss some important modifiers of this pathway. Nevertheless, the number of AHR modifier loci reported here is approximately 10-fold greater than what has been reported using mammalian cell culture and animal models .max was set at 2, 3, or 4, the resultant AHR–PIN was of a complexity that could not have resulted from random chance. Furthermore, the comparison of various simulations at different Dmax settings guided us to select the linking parameter at Dmax = 2. This setting of intervening links resulted in the highest level of statistical significance, displayed the lowest potential for false positive interactions, and decreased the map's visual complexity to a level that was readily understood in a two-dimensional map.Once we identified these AHR modifiers in yeast, we sought a way to position and characterize them in the context of the AHR pathway. Given the idea that PINs can be used to portray the cellular workings, we attempted to use our deletion data to generate and annotate an AHR–PIN . To consOur analysis of the AHR–PIN revealed an underlying modular structure. That is, there are areas in the AHR–PIN that display high interconnectedness of nodes, and these regions represent functionally related modifiers. The modularity of AHR–PIN was revealed by both computational and functional tests. In our initial computational approach, a total of ten clusters were identified, and the functional enrichment of each cluster was calculated by hypergeometric distribution .Although the computational approaches of module identification and annotation were useful in hypothesis generation, they did not provide a direct description of AHR signaling. Therefore, we set out to annotate the AHR–PIN with a number of functional tests. In our first annotation experiment (“domain influence”), we found that the AHR–PIN could be divided into three discrete functional modules . Additionally, each of these modules was found to overlap with one to several network clusters see . This tiA module that regulates AHR folding was identified by the known activities of its constituents, as well as the appearance of receptor aggregates when these modifiers were absent see . In addiThe AHR folding module can also be used to explain the existence of I-nodes within a functional module. Given their “linker” position and the observation that they often share similar annotated function with their neighboring M-nodes (data not shown), it is a logical prediction that I-nodes play a role in AHR signaling that is functionally similar to their modifier neighbors. We propose that I-nodes most commonly arise as the result of their essential gene nature  or because they represent a redundant gene product (white nodes in the figures). We offer two examples that support this idea. First, one essential gene I-node in the folding module, Cns1p, has recently been reported to be involved in AHR signaling . Second,The composition of the transcriptional activation module suggests that the AHR activates target genes via the coordination of histone acetylation, ATP-dependent chromatin remodeling, and direct recruitment of basal RNA polymerase II transcriptional apparatus see . We baseOur network analysis has also identified a functional module that regulates the ligand-dependent translocation of the AHR see . This nuA module that regulates the amount of receptor protein was also identified in our AHR–PIN see . This moA novel PASB-dependent step in AHR signaling appears to have been revealed by this network analysis see , group IWe began this study with the objective of defining the AHR signal transduction pathway in a manner that would allow us to quantify the number of loci and enumerate the steps involved in signaling. By integrating our deletion screen with the PIN framework and through subsequent computational and experimental annotations, we were able to identify modifier modules that regulate five distinct AHR signaling steps. In this regard, we found that the integration of multiple annotation approaches is vital for the reconstruction of the final picture by connecting and cross-validating individual information pieces. As interaction datasets become more fully developed and annotated, such a map will steadily improve and provide more accurate description of AHR signaling. Lastly, the systematic strategy that we developed in this work should be readily applicable to the study of most mammalian proteins to reconstruct corresponding modifier networks that regulate their signaling.S. cerevisiae strain BY4742  was used in this study. This deletion set was obtained from Research Genetics  in a 96-well arrayed format. The plasmid pCEN-AHR (PL1605) was constructed by replacing the TRP1 autotrophic marker of PL883 (HIS3 marker using a “marker swap” method (ADH1) promoter. LexA-AHRNΔ166 is a chimeric AHR, with its amino acid residues 1–166 replaced by residues 1–202 of bacterial repressor LexA, and is referred to in the Results section simply as “AHR” for convenience. The reporter plasmid pSH18–34 (PL623)  is a 2μ-based, URA3-selectable vector that contains the bacterial LacZ gene, as a reporter, under the control of eight LexA-binding sites. The plasmid pEG202  is a 2μ-based, HIS3-selectable plasmid containing the LexA1–202 sequence under the control of the ADH1 promoter. The plasmid pAHR (PL700) has been described previously (GAL4 inserted into the EcoRI site of pEG202. The control plasmid pAHRΔPASB (PL1799) is the same as pAHR except for the removal of the C-terminal half of the PAS domain. This pAHRΔPASB plasmid was constructed by subcloning the EcoRI fragment of PL248 . The OD600 was measured using a SpectraMax 250 microplate reader . Cells were then subjected to centrifugation at 3,500 rpm for 8 min, and the supernatant was decanted. The 96-well plates were placed upside-down on a stack of paper towels for 10 min to drain residual medium. For transformation, each plate was vortexed at maximal speed for 15 s before dispensing 22 μl of DNA in “OneStep” buffer  into each well. To make the DNA in “OneStep” buffer, one volume of DNA  was mixed vigorously by vortexing with ten volumes of “OneStep” buffer. After DNA was dispensed, the plate was quickly vortexed again at maximal speed for 10 s to resuspend the cells, followed by incubation at 45°C for 40 min. After this “heat shock” step, 5 μl of the transformation mix from each well was inoculated into a fresh 96-well flat-bottomed plate containing 96 μl per well of dropout medium without Trp, Ura, and His (dropout minus TUH medium) plus G418. The inoculum was gently mixed by vortexing and incubated at 30°C for about 4 d until transformants grew out.A high-throughput protocol was developed for 96-well transformation based on work previously described . Unless LacZ reporter assay, transformants from the 96-well plates were rearrayed into 384-well stock plates containing 30 μl per well of dropout minus TUH medium. The inoculum was incubated at 30°C for 2–3 d to allow cell growth. For the LacZ reporter assay at each agonist concentration, 0.5 μl of cell culture was transferred from the 384-well stock plate (30°C) into a clear-bottomed/black-walled 384-well assay plate  using a disposable 384-pin replicator (GenomeSystems/Incyte Genomics). In the 384-well assay plate, each well contained 18 μl of dropout minus TUH medium (diluted 1:4 in water) plus agonist at the tested concentration. The plates were then incubated at 30°C for 48 h to allow all strains to reach stationary phase. Cell growth was monitored by measuring the OD600 of each well using a SpectraMax Plus384 microplate reader (Molecular Devices). To initiate the fluorescence assay, 18 μl of lysis/assay buffer was added to each well. Lysis/assay buffer contained a mixture of CUG substrate , 10% SDS, 1 M NaPO4, and 25× TAE in the ratio of 1:1.4:350:17.5. For assays with pCEN-AHR transformants, no TAE was required. Plates were vortexed at medium speed for 1 min and left at room temperature for 20 min. The reaction was stopped by dispensing 6.5 μl of 25× TAE to each well and vortexing at medium speed for 1 min. The fluorescence emission of each well was detected using a Wallac “VICTOR V” microplate reader . The fluorescence reading was normalized to the corresponding OD600 value to obtain the LacZ reporter activity of each deletion strain.To perform the Selected deletion strains were transformed with the plasmid pAHRGFP. Transformants were incubated in a 96-well microtiter plate containing 100 μl per well of dropout minus TH medium at room temperature. Given that we have observed that small temperature shifts can affect AHR's localization, we found it more convenient to both grow and examine cells at the same temperature. For some samples, assays were repeated at 30°C using a heating chamber attached to the microscope. Such results were found to be comparable to those obtained at room temperature. For strains that reached early log phase, 0.5 μl of culture was mounted on a glass slide, and the AHR–GFP subcellular localization was examined using a Zeiss  Axiovert 200M microscope (α Plan-FLUAR 100× objective). Images were captured using an AxioCam HR digital microscope camera (Zeiss). To stain the nucleus in living cells, 4,6-diamidino-2-phenylindole (DAPI) was added to the dropout minus TH medium to a final concentration of 5 μg/ml.LacZ reporter activity of each deletion strain was compared to the average of wt BY4742 strain controls included in the same plate, and the fold change was obtained and log2 transformed. These data-processing steps, as well as subsequent modifier selection, were performed automatically using Perl scripts written “in house.” In brief, for the primary screen involving 4,507 deletion strains with low-copy pCEN-AHR system, a stringent cutoff of 4-fold change over wt control was chosen for selecting a pool of most significant AHR signaling mutants. This cutoff corresponds to a p value of less than 10–6 at all six assessed concentrations (null distribution: wt control). The initial positives were subject to validation and characterization in secondary screens with high-copy pAHR and control systems. The cutoffs for control pathways pGal4TAD and pAHRΔPASB in the secondary screens were chosen at 2-fold change over wt control, which corresponds to p values of 3.3 × 10–2 and 5.6 × 10–4 (null distribution: wt control), respectively.To identify deletions that modify AHR signaling, the http://dip.doe-mbi.ucla.edu) and the genetic interaction table from the MIPS database (http://mips.gsf.de/proj/yeast/). Perl scripts, written “in house,” were used to search the combined physical and genetic interaction database and identify all valid paths  that linked each pair of modifiers. The network graph was rendered using the Graphviz tool kit (http://www.research.att.com/sw/tools/graphviz/)  .Within experimental annotation layers of the AHR–PIN, the region corresponding to each functional module was outlined by a closed line (boundary) drawn manually on the network map. This boundary was delineated to include the maximal number of modifier nodes that are members of the corresponding functional module and the minimal number of modifier nodes that are nonmembers. This boundary was also defined in such a way that all enclosed modifier nodes were interconnected via paths within the enclosed region or through at most one modifier node outside. When defining functional modules in the summary AHR–PIN, the highest weight was given to the results from the localization influence experiments because these results provided the most direct indication of a modifier's effect on AHR signaling, and the lowest weight was given to the pharmacology clustering result because this result was highly sensitive to the choice of clustering algorithm.Table S1wt BY4742 strain. Also shown are their known gene names, products, gene descriptions, and Gene Ontology (GO) annotations .Click here for additional data file.Table S2This table contains all of the ORFs that were observed to influence the signaling of the AHR but not the pGal4TAD control. Also shown are their known gene names, products, gene descriptions, and GO annotations .(27 KB XLS).Click here for additional data file.Table S3This table summarizes the annotation on cellular functions of AHR modifiers. The annotation was derived from the YPD database, as of May 2002  .(23 KB XLS).Click here for additional data file.Table S4max = 2). Also shown are their known gene names, products, gene descriptions, and GO annotations .Click here for additional data file.Table S5p value is shown .This table summarizes the functional enrichment of each network cluster as calculated by the hypergeometric distribution of MIPS and GO annotations. For each cluster, the functional enrichment is determined by using M-nodes alone and both M- and I-nodes, respectively. In each case, the annotation that corresponds to the largest number of nodes in the cluster and the smallest (22 KB XLS).Click here for additional data file.


The 5T33 multiple myeloma is one of a series of transplantable murine myelomas arising spontaneously in C57BL/KaLwRij mice. This study describes the establishment and characterisation of the 5T33 murine myeloma in vitro as a cultured cell line in terms of its morphology, growth rate, expression of paraprotein (IgG2b) and tumorigenicity in syngeneic animals. The 5T33 cell line has been in continuous culture for over 10 months and has achieved more than passage 34. In culture, 5T33 myeloma grows as single cells or in small clusters of loosely adherent cells on an adherent stromal cell layer. Maximum doubling time is approximately 25 h, and over 90% of the cells express cytoplasmic IgG2b paraprotein. The cultured 5T33 myeloma cells are highly tumorigenic in C57BL/KaLwRij mice with as few as 500 cells inducing paralysis and death as early as day 36 post-tumour inoculation. Kinetics of tumour development and detection of IgG2b paraprotein are dose dependent. Two weeks following intravenous inoculation of 5 x 10(5) cultured 5T33 myeloma cells, tumour cells were readily identified in the bone marrow. By 3 weeks post-tumour inoculation, 5T33 myeloma cells were found in various tissues throughout the animal. Studies are now underway to determine the sensitivity of this cell line to various therapeutic modalities.


Our previous studies demonstrated that, in other cell types, galangin is a potent inhibitor of the aryl hydrocarbon receptor (AhR), an environmental carcinogen-responsive transcription factor implicated in mammary tumor initiation and growth control. Because some current breast cancer therapeutics are ineffective in estrogen receptor (ER) negative tumors and since the AhR may be involved in breast cancer proliferation, the effects of galangin on the proliferation of an ERFhAhRR) were studied in Hs578T cells by western blotting for nuclear  AhR and by transfection of an AhR-driven reporter construct, pGudLuc. The effects of these agents on cell proliferation were studied by 3H-thymidine incorporation and by flow cytometry. The effects on cyclins implicated in mammary tumorigenesis were evaluated by western blotting.AhR expression and function in the presence or absence of galangin, a second AhR inhibitor, α-naphthoflavone (α-NF), an AhR agonist, indole-3-carbinol, and a transfected AhR repressor-encoding plasmid . Galangin inhibited transition of cells from the G0/G1 to the S phases of cell growth, likely through the nearly total elimination of cyclin D3. Expression of cyclins A and E was also suppressed.Hs578T cells were shown to express high levels of constitutively active AhR. Constitutive and environmental chemical-induced AhR activity was profoundly suppressed by galangin as was cell proliferation. However, the failure of α-NF or Galangin is a strong inhibitor of Hs578T cell proliferation that likely mediates this effect through a relatively unique mechanism, suppression of cyclin D3, and not through the AhR. The results suggest that this non-toxic bioflavonoid may be useful as a chemotherapeutic, particularly in combination with agents that target other components of the tumor cell cycle and in situations where estrogen receptor-specific therapeutics are ineffective. Flavonoids are a diverse class of naturally occurring polyphenolic plant compounds that have a variety of therapeutically important biological activities. Several thousand plant flavonoids have been identified and biologically significant levels of bioflavonoids are consumed about 1 g/day) by humans living in Western cultures  g/day by, flavonoAlpinia officinarum, a plant used for many years in Asia as an herbal therapeutic -thymidine incorporation experiments, cell cycle and apoptosis analyses, and western immunoblotting, participated in the experimental design and coordination of this project, and participated in writing the manuscript. XY performed transient transfections and reporter assays and western immunoblotting, participated in the experimental design and coordination of the project, and contributed to the writing of the manuscript. DHS conceived of the experimental design, performed statistical analyses, and prepared the manuscript. All authors approved the final manuscript.TJM performed [


Cell killing can be achieved in an acidic environment in tissue culture (medium pH less than 7.0) by agents ) which transport protons from the extracellular space into the cytoplasm. Cell killing is enhanced when these agents are used in combination with compounds ) which inhibit the membrane-based exchangers responsible for the regulation of intracellular pH (pHi). We describe experiments which assess the ability of these agents to kill tumour cells in spheroids and in vivo. Both nigericin and CCCP were observed to penetrate tissue based on their ability to kill tumour cells in spheroids. The mean extracellular pH (pHe) of the KHT fibrosarcoma and the EMT-6 sarcoma were observed to be 0.21 and 0.32 pH units more acidic than the mean pHe in muscle tissue. Intraperitoneal (i.p.) administration of the vasodilator hydralazine (10 mg kg-1) caused a reduction of the mean pHe of the KHT but not the EMT-6 tumour. Nigericin  plus amiloride  followed 30 min later by hydralazine  reduced the surviving fraction of cells in the KHT and EMT-6 tumours, but had minimal effects on growth delay. When KHT tumours were treated with 15 Gy X-rays followed immediately by nigericin plus amiloride and hydralazine a reduced surviving fraction as well as an increase in tumour growth delay was observed compared to radiation alone. The administration of nigericin  or the combination of nigericin  followed by hydralazine ) resulted in reductions of tumour pHi of 0.27 and 0.29 pH units respectively as determined by 31P magnetic resonance spectroscopy (MRS). Our results show that the combination of nigericin and hydralazine (with or without amiloride) can kill cells in rodent solid tumours and that cell killing is associated with a reduction in the mean pHi of tumour cells.


The effects of cholera toxin (CT) and 8-chloro-cAMP (8-Cl-cAMP) on cell growth were investigated using two human pancreatic carcinoma cell lines . CT, which catalyses the ADP ribosylation of Gs, suppresses the proliferation of MIA PaCa-2(PC) cells. CT at the low dose of 0.1 pg ml-1 was inhibitory of PC cell growth, and the maximum suppression (70%) was achieved at a CT concentration of 100 pg ml-1. This phenomenon was reversible. The production of cAMP by CT (100 pg ml-1) in PC cells was enhanced 320-fold compared with the control. In addition, cAMP analogues  and forskolin decreased the growth rate of PC cells in a dose-dependent manner. These results support the view that CT suppresses PC cell growth by stimulating cAMP production. Conversely, Panc-1 cells were far less sensitive to CT in cell growth and cAMP production. 8-Cl-cAMP was also less effective on Panc-1 cell growth. The binding of an insulin-like growth factor (IGF)-I and transforming growth factor (TGF)-alpha, which has been shown to stimulate PC cell growth in an autocrine manner, to PC cells was not modified in cells treated with CT or 8-Cl-cAMP. The results suggest that the inhibitory actions of these substances do not occur at the level of the receptor for IGF-I or EGF/TGF-alpha. We have previously shown that phorbol esters, which decrease the binding of TGF-alpha to PC cells, has an anti-proliferative activity on these tumour cells. Inhibited cell growth by maximum suppressive dose of CT or 8-Cl-cAMP was further inhibited by TPA. In addition, an oncogene product of K-ras which is commonly activated in pancreatic cancer, was increased by CT and 8-Cl-cAMP. It is concluded that CT and 8-Cl-cAMP inhibit PC cell growth, presumably in a similar manner, and their mechanism(s) of action may be different from that of TPA. The anti-proliferative effect of CT or 8-Cl-cAMP was enhanced by TPA, implying that the combination of these substances results in increased inhibition of the PC cell growth.


Dioxins and related compounds are suspected of causing neurological disruption in human and experimental animal offspring following perinatal exposure during development and growth. The molecular mechanism(s) of the actions in the brain, however, have not been fully investigated. A major participant in the process of the dioxin-toxicity is the dioxin receptor, namely the aryl hydrocarbon receptor (AhR). AhR regulates the transcription of diverse genes through binding to the xenobiotic-responsive element (XRE). Since the AhR has also been detected in various regions of the brain, the AhR may play a key role in the developmental neurotoxicity of dioxins. This study focused on the effect of AhR activation in the developing neuron.The influence of the AhR on the developing neuron was assessed using the Neuro2a-AhR transfectant. The undifferentiated murine neuroblastoma Neuro2a cell line (ATCC) was stably transfected with AhR cDNA and the established cell line was named N2a-Rα. The activation of exogenous AhR in N2a-Rα cells was confirmed using RNAi, with si-AhR suppressing the expression of exogenous AhR. The neurological properties of N2a-Rα based on AhR activation were evaluated by immunohistochemical analysis of cytoskeletal molecules and by RT-PCR analysis of mRNA expression of neurotransmitter-production related molecules, such as tyrosine hydroxylase (TH).N2a-Rα cells exhibited constant activation of the exogenous AhR. CYP1A1, a typical XRE-regulated gene, mRNA was induced without the application of ligand to the culture medium. N2a-Rα cells exhibited two significant functional features. Morphologically, N2a-Rα cells bore spontaneous neurites exhibiting axon-like properties with the localization of NF-H. In addition, cdc42 expression was increased in comparison to the control cell line. The other is the catecholaminergic neuron-like property. N2a-Rα cells expressed tyrosine hydroxylase (TH) mRNA as a functional marker of catecholaminergic neurotransmitter production. Thus, exogenous AhR induced catecholaminergic differentiation in N2a-Rα cells.The excessive activation of AhR resulted in neural differentiation of Neuro2a cells. This result revealed that dioxins may affect the nervous system through the AhR-signaling pathway. Activated AhR may disrupt the strictly regulated brain formation with irregular differentiation occurring rather than cell death. AhR in (TCDD) ,4. Severn (TCDD) ,6.The AhR is activated by binding to ligands, including dioxins, translocates from the cytoplasm to the nucleus and then binds to the consensus sequence known as XRE (xenobiotic responsive element) . XREs arOne of the dioxins, TCDD, can also influence the developing brain. After exposure to experimental animals, TCDD was detected in the brain and impaired their learning behavior. Perinatal exposure to TCDD has also been found to remarkably affect learning ability in rats and monkeys -17. HoweThe dioxin receptor, namely the AhR, is expressed in various regions of the brain ,19 durinin vivo [The regulation of neurogenesis is key event for controlling brain development. To understand the regulatory mechanisms of neurogenesis, neuroblastoma is widely used as an experimental model. Neuroblastoma usually arises from the proliferation of neural crest-derived precursors resulting in defective differentiation in vivo . Neuroblin vivo ,25. Neurin vivo ,27. In t2/95% air.Neuro2a, a murine neuroblastoma cell line, was purchased from American Type Culture Collection, and was routinely grown in Dulbecco's modified Eagle's medium and medium F12 in a 1:1 ratio, supplemented with 10% fetal calf serum. Cells were grown at 37°C in a humidified atmosphere of 5% COPyrobest DNA polymerase . PCR primers were designed to contain appropriate restriction sites for subcloning. The sequences of the primers were: 5'-CCCAAGCTTACCATGAGCAGCGGCGCCAACATCA-3' (HindIII) and 5'-CCGCTCGAGAGGAATCCGCTGGGTGTGATATCAG-3' (XhoI). After digestion with HindIII and XhoI, the PCR product was cloned into the mammalian expression vector pcDNA4/V5-His  digested with HindIII and XhoI. The resulting vector contained the coding sequence of AhR fused to the V5/His tag under the control of cytomegalovirus promoter. Correct insertion of the PCR product was verified by DNA sequencing.The coding sequence of AhR was obtained by RT-PCR from rat brain RNA with 4) were transfected with 0.8 μg of constructed plasmid using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen Japan KK). Cells transfected with a vector without the insert were used as a control. After culturing for 72 h in normal medium, the cells were switched to medium supplemented with 250 μg/ml antibiotic zeocin (Invitrogen Japan KK). The medium was changed every 3 days during selection of viable cells. After 2 weeks, viable cells were isolated using cloning rings. The isolated clones were cultured in medium supplemented with 250 μg/ml zeocin for another 2 weeks and the viable clones were selected for further characterization.Neuro2a cells were maintained and grown in Dulbecco's modified Eagle's medium and medium F12 in a 1:1 ratio, supplemented with 10% fetal calf serum. The cells (8 × 10Ex Taq polymerase (TAKARA BIO Inc.) using gene-specific primers. The primer sequences were as follows: AhR, 5'-CCGTCCATCCTGGAAATTCGAACC -3', 5'-CCTTCTTCATCCGTTAGCGGTCTC; CYP1A1, 5'-TTGCCCTTCATTGGTCACAT-3', 5'-GAGCAGCTCTTGGTCATCAT-3'; AhRR, 5'-CCCAAACTCAATTACTTAGCAGG-3', 5'-GCTTCCAGGGTAGGGCACACAGG-3'; GST, 5'-CCTGGCTGCAGCAGGGGTGGAG-3', 5'-CGGTTTTTGGTCCTGTCTTTTGC-3'; cdc42, 5'-GATACTGCAGGGCAAGAGGA -3', 5'-CAGGCACCCACTTTTCTTTC-3'; TH, 5'-CCACGGTGTACTGGTTCACT-3', 5'-GGCATAGTTCCTGAGCTTGT -3'; Nurr, 5'-CCAATCCGGCAATGACCAG-3', 5'-GTCAGCAAAGCCAGGGATCTTC-3'; Cyclophilin, 5'-GTCTCCTTCGAGCTGTTTGCAG-3', 5'-CCATCCAGCCATTCAGTCTTGG-3'. PCR products were electrophoresed on 3% Nusieve 3:1 agarose gel (TAKARABIO inc.) and then stained with ethidium bromide. The intensities ofthe bands were quantified using ImageJ software [Total RNA was isolated from cultured cells following a 2-day culture using RNeasy . Rat brain RNA was extracted from 8-week-old Sprague-Dawley rats. The rats were killed by cervical dislocation under ether anesthesia and the brains were dissected.Total RNA was extracted by Quick Prep Total RNA Extraction Kit . Total RNA of mouse brains were purchased from ZYAGEN laboratories . These RNAs were reverse-transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen Japan KK). PCR was performed with software  and thenTwo siRNAs, si-AhR and scramble, were synthesized (Qiagen KK) and transfected into the cells using RNAiFect according to the manufacturer's instructions (Qiagen KK). The siRNA sequences were: si-AhR, 5'-UCCCACAUCCGCAUGAUUATT-3', 5'-TTAGGGUGUAGGCGUACUAAU-3'; scramble, 5'-UACCCAUCAUGACCCUGAUTT-3', 5'-TTAUGGGUAGUACUGGGACUA-3'. Two days after transfection, total RNA was extracted from the cells and reverse-transcribed with Superscript II reverse transcriptase. The cDNAs were used in real-time PCR as templates for quantification.AhR, CYP1A1 and cdc42 mRNA expression were quantified in AhR by real-time PCR. The primers used in real-time PCR were the same as those described above. Real-time PCR was carried out with DNA engine  using the DyNAamo SYBR Green qPCR Kit . The PCR conditions were: 95°C for 5 min, 40 cycles of 95°C for 10 s, 58°C for 10 s, and 72°C for 20 s with fluorescence measurements read during each cycle. The expression levels of AhR and CYP1A1 mRNAs were normalized to those of cyclophilin mRNA. The series of measurements was performed four times.Tissues and cells were homogenized in phosphate buffered saline (PBS) and denatured with SDS. Total protein concentrations were determined by Bio-Rad Protein Assay . Proteins (30 μg/lane) were fractionated on 10% SDS-PAGE and transferred to PVDF membrane . Membranes were blocked with Block Ace  and then incubated with an anti-AhR mouse monoclonal antibody . After incubation with the primary antibody, the membrane was washed three times with tris-buffered saline (TBS). The blots were subsequently exposed to an alkaline phosphatase-conjugated rabbit anti-mouse IgG . The blots were visualized with BCIP-NBT substrate solution .After being cultured for 2 days, the cells were rinsed with phosphate-buffered saline (PBS) and fixed with methanol for 25 min at 4°C. They were incubated with monoclonal anti-phosphorylated neurofilament 200-kDa antibody . After washing five times with PBS, the cells were treated with FITC-conjugated goat anti-mouse IgG  and were then subjected to fluorescence microscopy.3) were seeded in each well of a 96-well flat bottom culture plate and cultured for 1, 3 and 7 days. Cell proliferation was measured by Alamar Blue dye reduction assay . Briefly, Alamar Blue solution  was added to each well and the plate was incubated for 2h at 37°C. Dye reduction was measured in a Mithras LB940 microplate reader  at 530 nm excitation and at 590 nm emission. Cell proliferation rate was calculated as the fold increases, compared with the value at day 1.Cells . All experiments were replicated at least three times. Data were analysed using Student's T-test with the Exel software package.To investigate the function of AhR in developmental neurotoxicity, we attempted to establish Neuro2a-AhR transfectants. The murine neuroblastoma cell line, Neuro2a, was transfected with AhR cDNA in the expression vector (pcDNA4/V5-His), which includes the V5 epitope and the cytomegalovirus promoter. Seventy-five stably transfected clones were obtained after selection with zeocin. One of the clones, named N2a-Rα, was selected and used in a further series of experiments. Figure RT-PCR analysis was performed using mRNA isolated from transfectants to evaluate the expression of AhR mRNA with AhR-specific primers. The AhR primers were designed for rat AhR, but also specifically recognize and amplify mouse AhR under identical PCR conditions Fig . AhR mRNIn order to confirm the transcriptional activity of the exogenous AhR in N2a-Rα, we analyzed CYP1A1, GST and AhRR (AhR repressor) mRNA expression as functional markers of AhR -11,29. TIf the AhR stimulated the expression of XRE-mediated genes in N2a-Rα, such as CYP1A1, this expression would be attenuated by suppressing AhR activity. In this context, we attempted to suppress the AhR transcription using RNA interference. Two siRNAs  were transfected into N2a-Rα cells. Two days after transfection, the expression of AhR and CYP1A1 mRNA was measured by real-time quantitative PCR. As a result, AhR mRNA was decreased about 30% in N2a-Rα cells transfected with AhR siRNA, while the CYP1A1 mRNA level was decreased about 60%  and Nurr-1. TH is the rate-limiting enzyme in the production of neurotransmitters and is considered an adequate marker of catecholaminergic neurons ,41. NurrActivation of the AhR by dioxins results in the induction of xenobiotic metabolism and successive toxic responses, including hepatocellular damage, immune system disorders, reproductive system disorders, teratogenesis and carcinogenesis -48. In tIn the nervous system, dioxins and related compounds are suspected to cause neurological disruption in human and experimental animal offspring following perinatal exposure during development and growth ,53. The AhR-mediated pathways exist to mediate the effects of TCDD in many organs such as the liver, reproductive organs or immune tissues. ,6. HowevWe have shown that the AhR is also functional in N2aRα cells even in the absence of ligand. This is based on the finding that the increase of CYP1A1 mRNA expression caused by over-expression of exogenous AhR was clearly suppressed by repression of the AhR with siRNA. There have been several other examples in which transfected AhR has been found to be active in the host cells ,57, althThe possible underlying mechanisms of AhR responses have been discussed previously. In many cell models, the AhR has been shown to stimulate growth arrest and apoptosis . For exaDespite the fact that little evidence supporting the physiological function of the AhR in neural cells has been found so far, it has been reported that AhR is a member of the bHLH family transcription factor family. Although the AhR is an orphan receptor whose function has not been revealed, it is helpful for understanding AhR function to consider the relationships between AhR and other members of the bHLH family. Members of the bHLH family form a molecular-signaling network related to neural development and differentiation. For example, Hes-1 (the Hairy and Enhancer of Split homolog-1) and Hes-5 down-regulate Mash-1 and Math-1, respectively. Mash-1 and Math-1 subsequently down-regulate NeuroD and Math-2, and finally regulate the expression of nerve-specific genes . In thisConsequently, in developing neurons, irregular or excessive activation of AhR possibly causes disrupted neural development rather than cell death. In the brain, TCDD exposure during the perinatal period may affect XRE-regulated gene expression through AhR activation and disrupt the differentiation of neurons, which are strictly regulated for developing brain formationIn this study, we have shown that over-expression of AhR caused neural differentiation of Neuro2a cells. Neruo2a cells transfected with AhR, named N2a-Rα, may mimic the neurons forming the neural network during the perinatal stages, expressing AhR in the presence of the ligand. The AhR is a major participant in the expression of neurotoxicity and plays a role in the structural and functional development of neural cells. We hypothesized that perinatal exposure to dioxins may disrupt the strict regulation of brain formation with irregular differentiation rather than cell death. In consequence, the decline of neural function based on excess differentiation of neurons by AhR activation may play an important role in the developmental neurotoxicity of dioxins.AhR, aryl hydrocarbon receptorXRE, xenobiotic responsive elementTH, tyrosine hydroxylasebHLH-PAS, basic helix-loop-helix/Per-Arnt-Simp-dioxinTCDD, 2, 3, 7, 8, -tetrachlorodibezo-CYP, cytochrome P-450FITC, fluorescein isothiocyanateThe author(s) declare that they have no competing interests.EA designed the study and performed all experiments. SY was responsible for the cloning and maintenance of all cell strains constructed. MIS collaborated on experimental design, participated in data analysis, and wrote the manuscript. All authors read and approved the final manuscript.


Drosophila. Flies lacking the DJ-1 gene showed selective sensitivity to widely used agricultural toxicants that kill neurons mainly through oxidative stress. The studies, published 6 September 2005 in Current Biology, suggest that normally “DJ-1 has a neuroprotective role against different oxidative stimuli,” says Darren Moore, an instructor in neurology at the Johns Hopkins University School of Medicine who has studied the gene. However, if DJ-1 stops working—because of either an inherited mutation or toxicant exposure—oxidative stress may wreak havoc on the brain, killing dopamine-producing neurons.A gene linked to familial Parkinson disease (PD) may protect neurons from oxidative damage, according to two independent studies in the fruit fly DJ-1 mutations may sensitize cells to the harmful effects of oxidative stress. This type of oxidative damage happens when unstable oxygen molecules react with certain components inside cells in a manner similar to the process that converts iron to rust. Many environmental insults—including exposure to certain agricultural chemicals—can generate these unstable oxygen molecules.Experiments in cultured cells and in knockout mice have hinted that DJ-1 interacts with such oxidative stress agents, Nancy Bonini, a professor of biology at the University of Pennsylvania, Philadelphia, and her colleagues first identified DJ-1 in Drosophila, which they found exists in two forms: DJ-1α (expressed primarily in the testes) and DJ-1β (expressed everywhere). They then created a line of Drosophila mutants completely lacking both forms.To pin down how DJ-1 had normal life spans and showed no neuronal degeneration. However, when Bonini and her colleagues exposed flies to the herbicide paraquat, DJ-1 mutants died much sooner than normal flies. The mutants also showed marked sensitivity to the insecticide rotenone and to hydrogen peroxide—both agents that promote oxidative stress. These results suggest that DJ-1 normally protects against oxidative stress and that its inactivation may leave neurons susceptible to oxidative damage. The team also found that exposure to paraquat led to biochemical modification of the DJ-1β protein, a change Bonini says may somehow influence the ability of DJ-1 to protect neurons from oxidative damage.The flies with no Drosophila DJ-1 mutant. They disrupted the function of DJ-1β by inserting a mutation into the middle of the gene. Surprisingly, Min says, they found that dopamin-ergic neurons in these mutants survived longer into old age than did neurons of normal flies. They also found that their DJ-1β mutants were much more resistant to paraquat insult than were normal flies.In the other paper, Kyung-Tai Min, an investigator at the National Institute of Neurological Disorders and Stroke, and his colleagues examined a different type of DJ-1α expression—the loss of DJ-1β somehow encouraged a compensatory upregulation of DJ-1α, which the authors believe protected the fly from paraquat-induced oxidative damage. When they treated the same flies with hydrogen peroxide, however, they found that the DJ-1β mutants were extremely susceptible to early death. Min says this suggests that DJ-1α and DJ-1β may normally protect cells against different types of agents that promote oxidative stress.Further examination revealed that these flies had elevated DJ-1 fly strains will probably make an ideal model for PD, according to Moore, because the flies don’t suffer from the neurodegeneration seen in humans with DJ-1 mutations. Nevertheless, says Bonini, studies of DJ-1 in Drosophila will provide greater understanding of fundamental activities of the gene, helping to elucidate how its function may be critical in PD.Neither of the mutant


Evidence was obtained showing that GSH protects against the cytotoxicity of 4-hydroperoxycyclophosphamide (4-OOH-CP) by minimizing the spontaneous fission of 4-hydroxycyclophosphamide (4-OH-CP), its breakdown product, to the ultimate toxic species, phosphoramide mustard (PM). This conclusion was borne out in two series of experiments. The first demonstrated that 4-OH-CP was progressively more stable in aqueous solutions containing increasing concentrations of GSH. The second series of experiments were carried out with tumour cell lines with high (SKOV-3) and low (KHT) GSH contents. The cytotoxicity of 4-OOH-CP, a stable precursor that rapidly gives rise to 4-OH-CP spontaneously under physiological conditions, was enhanced in GSH-depleted SKOV-3 cells, but was unchanged in GSH-depleted KHT cells. It is concluded that the high GSH content of SKOV-3 cells provides a significant protection against 4-OH-CP by limiting the breakdown/activation of 4-OH-CP. Deschloro-4-hydroperoxycyclophosphamide (deschloro-4-OOH-CP), an analogue of 4-OOH-CP that generates acrolein (AC) but not PM in the spontaneous fission reaction, is essentially non-toxic when compared with 4-OOH-CP but is equally potent in depleting GSH. It is postulated that AC may promote the cytotoxicity of the parent 4-OH-CP by depleting cellular GSH. Consequently, the stabilising influence of GSH on 4-OH-CP is removed, leading to increased formation of PM, the ultimate cytotoxic agent.


The relationship between colony numbers and concentration of cells plated is an important parameter of clonogenic assay systems. The cloning efficiency for an ideal sample should be independent of cell concentration, thus giving a straight line through the origin when colony numbers are plotted against cell concentration. A simple statistical method has been developed to test if this is the case for individual tumour samples. Colony data from 51 freshly obtained tumour samples, which had sufficient cells to plate 3 or more dilutions and gave at least 20 colonies per plate at one or more of the dilutions, were tested. The results indicated that colony formation was linear for 27 (53%) of the samples. The remaining 24 samples could be classified into 2 groups: type I, in which cloning efficiencies increased with increasing cell concentration and type II, which had reduced cloning efficiencies at high cell concentrations. Fifteen (29%) of the samples had type I non-linearity and 9 (18%) exhibited non-linearity of type II. These findings indicate that the relationship between colonies and cells plated should be examined for each biopsy sample particularly in each experiment where the effects of cytotoxic drugs are tested.


Interestingly, DJ-1 expression was increased in brains of zebrafish under conditions of oxidative stress, indicating that DJ-1 is a part of stress-responsive machinery. Since oxidative stress is one of the major contributors to the development of Alzheimer's disease (AD), we also examined DJ-1 expression in AD brains. Using DJ-1 specific antibodies, we failed to detect a robust staining of DJ-1 in brain tissues from control subjects. However, DJ-1 immunoreactivity was detected in hippocampal pyramidal neurons and astrocytes of AD brains. Therefore, our results strongly suggest that DJ-1 expression is not necessary during zebrafish development but can be induced in zebrafish exposed to oxidative stress and is present in human AD brains.Mutations in the DJ-1 gene have been linked to autosomal recessive familial Parkinson's disease. To understand the function of DJ-1, we determined the DJ-1 expression in both zebrafish and post mortem human brains. We found that DJ-1 was expressed early during zebrafish development and throughout adulthood. Knock down (KD) of DJ-1 by injection of morpholino did not cause dramatic morphologic alterations during development, and no loss of dopaminergic neurons was observed in embryos lacking DJ-1. However, DJ-1 KD embryos were more susceptible to programmed cell death. While a slight reduction in staining for islet-1 positive neurons was observed in both DJ-1 KD and H Parkinson's disease (PD) and Alzheimer's disease (AD) are the two most common neurodegenerative disorders. PD is characterized by loss of dopaminergic (DA) neurons in the substantia nigra and accumulation of intraneuronal inclusions known as Lewy bodies. AD is characterized by two major hallmarks, neurofibrillary tangles and neuritic plaques. Genetic studies of PD have demonstrated that two autosomal dominant genes (α-synuclein and LRRK2) -3 and thin situ hybridization [2O2 significantly increases the immunoreactivity of DJ-1 [High levels of DJ-1 mRNA have been detected in neuronal and non-neuronal populations of several regions in mouse brain by dization . A recen of DJ-1 . This up of DJ-1 .Pharmacological studies have shown that DJ-1 is needed for the protective effects of certain compounds which are known to inhibit the production of reactive oxygen species . DJ-1 prL166P in addition to E64D, M26I, A104T, and D149A is one of known mutations within DJ-1 that has been identified from familial PD cases. These mutant forms of DJ-1 show different levels of structural alteration (except for E64D due to similar residue replacement), which lead to reduced stability of DJ-1 ,21. The 2O2, and DJ-1 appeared to function as a peroxiredoxin-like peroxidase in these animals [DJ-1 knockout (KO) mice do not differ from wild type mice with respect to morphology or dopaminergic cell loss -24, with animals . In zebr animals , and KD  animals . In Dros animals .Zebrafish are beginning to be used for aging research, e.g., brief exposure of zebrafish embryos with Dichlorodihydrofluorescein diacetate allows repeated measurement of the level of reactive oxygen species generation in live fish . For neu2O2. We found that KD of DJ-1 and H2O2 had similar effects on islet-1 positive neurons  homeodomain protein), with both groups of embryos showing an increased number of apoptotic cells. Since the oxidative stress is one of the major contributors to the AD pathogenesis [In this report, we compared KD of DJ-1 to oxidative stress in zebrafish embryos. In addition, we examined DJ-1 expression in the adult fish brain by treating live animals with Hogenesis ,39, we eEmbryos were obtained from natural spawning of wild-type (Tubingen longfin strain) adults and were raised and staged according to Kimmel et al. .DJ-1 MO, directed against the 5' untranslated region (UTR) of DJ-1  was injected into fertilized zebrafish eggs at the one-cell stage at which time morpholinos can rapidly spread into all cells. The morpholino sequences were as follows: DJ-1 MO, 5'-GCGTCTAATAACCTGTGCGTGTCTG-3' control MO, 5'-CCTCCTACCTCAGTTACAATTTATA-3'.The above standard control MO was synthesized against the zebrafish β-globin intron. MO oligonucleotides were dissolved in 1× Danieu solution at 5 mM, diluted to 0.1 mM prior to injection, and ~4 ng was injected into each embryo. After injection, embryos were incubated at 28.5°C in embryo medium  containiTUNEL assay was performed using the TMR-RED in situ cell death detection kit  accordinIn situ hybridization was carried out according to standard protocols  using a Human postmortem cortical tissue was collected in accordance with Institutional Review Board-approved guidelines at Brigham and Women's hospital. Blocks of cortex were fixed for ~2 hrs in 10% neutral buffered formalin, as previously described . ParaffiHuman, mouse and zebrafish brain samples were homogenized in lysis buffer containing 0.2% NP-40. The lysates were collected after 1 hr of centrifugation at 100 000 × g (human and mouse) or after 5 min of centrifuge at 18,000 × g (zebrafish). Individual de-chorionated and de-yolked embryos were lysed, and all samples were heated in 1 × sample buffer  and then separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Antibodies KAM-SA100  and anti-DJ-1-N (1:500) and an enhanced chemiluminescence system  were used to detect the western blots.Wildtype CHO cells and APP stably transfected CHO cells (CHO+APP) were grown for 24 hrs before the conditioned media were collected for the measurement of Aβ levels by ELISA, as previously described . The samWe generated an affinity purified polyclonal antibody DJ-1-N against the N-terminus of DJ-1 that recognizes a single band at 23 kDa corresponding to endogenous DJ-1 from human cortex homogenate; this band was absent when the antibody was preabsorbed with its cognate peptide Fig. . The samUsing the human DJ-1 sequence, we identified multiple zebrafish ESTs and obtained the complete zebrafish DJ-1 sequence by the alignment of these ESTs with genomic zebrafish DJ-1 sequences from the Sanger Institute. We only found one DJ-1 homologue in zebrafish. Zebrafish DJ-1 has 4 exons that encode 189 amino acids, and the protein is 87% homologous to human DJ-1. cDNA from zebrafish embryos at the 1-somite stage was prepared and the full-length zebrafish DJ-1 cloned. The sequence of cDNA was confirmed and found to be identical to the one previously reported .A morphant technology that uses morpholino phosphodiamidate anti-sense oligonucleotides  to target genes in vivo -49 was uBoth control and DJ-1 MO injected embryos shared an almost identical morphology Fig. , and no 2O2 to induce an environment of oxidative stress. We found that control MO injected embryos showed well defined islet-1 positive neurons along the spinal cord  also showed DJ-1 immunoreactivity in scattered astrocytes (data not shown), but they showed weak or no DJ-1 staining in pyramidal neurons of the CA1, CA2, CA3 sub-regions and the hilus of the dentate gyrus (CA4) Fig. , except To search for any mechanistic cause for DJ-1 expression in AD brains, we examined the effect of Aβ on the regulation of DJ-1 expression. We have used two cell lines, CHO that expresses endogenous levels of APP, and CHO+APP that expresses high levels of APP and generates a large amount of Aβ. When we measured the conditioned media from CHO and CHO+APP cells, we found elevated levels of Aβ40 Fig.  and 7B. It is known that oxidative stress and mitochondrial dysfunction contribute to the pathogenesis of AD and PD. In AD, Aβ may directly interact with mitochondria and lead to increased free radical production; in PD, DJ-1, parkin and PINK1 are all linked to oxidative stress and/or mitochondrial dysfunction {Reviewed by ,53}. In Consistent with earlier studies in DJ-1 KO mice -24, KD oWe found that DJ-1 KD embryos have an increased number of apoptotic cells, consistent with an earlier report showing a p53 dependent apoptosis in DJ-1 KD zebrafish embryos . In cult2O2 on DJ-1 expression in the brains of adult zebrafish in vivo. For the first time, we found an increase in DJ-1 protein in brains of adult fish subjected to a stressful environment such as oxidative stress. While it is difficult to discern whether the increase in DJ-1 was derived from neuronal or glial sources, we predict that it was a global response from exposure to H2O2 in the environment.Recent studies have shown that DJ-1 expression is up-regulated in cultured cells in an environment that promotes the formation of intracellular reactive oxygen species ,14. We hTo further address this relationship, we utilized brain sections from post mortem AD and control subjects. We found that the avidity of different antibodies and the methods for preserving brain tissues were critical for revealing DJ-1 immunostaining. Whereas antibodies raised against the C-terminus of DJ-1 Park7) ,9 or ful ,9 or fuOur comparison staining with DJ-1-N and other DJ-1 antibodies  reveals an important finding on the potential binding region of DJ-1 when it dimerizes. Earlier reports, including ours, have demonstrated that DJ-1 dimerizes and forms a high molecular weight complex in cultured cells and human brains ,59. It s2O2. Therefore, the similarity of increased DJ-1 staining in H2O2 treated zebrafish and AD brains suggest that a common pathway might be elicited under damaging conditions. As previously reported, DJ-1 is needed to avoid oxidative stress-induced cell death in cultured neuroblastomas, dopaminergic cells and primary neuronal cells [The intriguing finding of positive staining of DJ-1 in AD brains but not control brains has important implications for both PD and AD pathogenesis. In brains of AD patients, the neuritic plaque composed of Aβ is one of the major characteristics of AD pathology. A lack of change in DJ-1 expression in cultured cells exposed to high levels of Aβ suggests that Aβ has no direct effect on steady state levels of DJ-1. During the neurodegenerative process, accumulation of reactive oxygen species renders neurons, such as pyramidal neurons around hippocampal area, extremely vulnerable, and gradual neuronal loss is inevitable when the brain is exposed to insults. The fact that DJ-1 itself is subjected to cysteine and methionine oxidation  as well al cells . Therefo2O2 [Furthermore, DJ-1 is a non-traditional peroxiredoxin-like peroxidase , and enh2O2 .2O2 illustrates a new plate form that allows us to examine the role of DJ-1 involved in response to oxidative stress. Investigation of the function/dysfunction of DJ-1 in a whole vertebrate animal exposed to a stressful environment has a direct physiological relevance to human diseases. Therefore, exploring mechanisms of DJ-1 associated with PD and AD pathogenesis will provide insight into common pathways involved in both neurodegenerative diseases. Any newly identified pathways will offer novel drug targets and exciting opportunities for therapeutic intervention.Future studies are necessary to characterize the role of DJ-1 in PD and AD pathogenesis such as whether the DJ-1 protein we found in pyramidal neurons in AD hippocampus is an acidic isoform , basic iAD: Alzheimer's disease; Aβ: amyloid β-protein; DA: dopaminergic; hpf: hours post fertilization; KD: knock down; KO: knock out; MO: morpholino; MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PD: Parkinson's disease; TH: tyrosine hydroxylase.The authors declare that they have no competing interests.SB and JS carried out the biochemical and immunohistochemical analyses, HL carried out zebrafish experiments, TY carried out cell culture experiments, MS, JS, MGS, CAL, and QL participated in the design of the study, SB and WX conceived of the study and draft the manuscript. All authors read and approved the final manuscript.