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) (wt strain (To accomplish this screen, we employed the yeast deletion strains made available by the  Project . We devea (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 (1.3 and 1.2 for Dmax = 3 and Dmax = 4, respectively). 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 (MATα, his3Δ1, leu2Δ0, lys2Δ0, ura3Δ0) 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 (V1M LiAc:V50% PEG 3350 = 1:4, with BME added to 0.77% V before use) into each well. To make the DNA in “OneStep” buffer, one volume of DNA (5 μg/μl ssDNA, 0.1 μg/μl each plasmid 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 (#F-2905, Molecular Probes, Eugene, Oregon, United States), 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 (Perkin-Elmer, Boston, Massachusetts, United States). 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 (Oberkochen, Germany) 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.


Vertebrates come in a dazzling array of shapes and sizes, from blue whales to pygmy bats, their overt morphology determined largely by the skeleton. The head skeleton in particular has undergone remarkable diversification, as is beautifully illustrated in Darwin's examination of beak morphology in Galapagos finches. It is now appreciated that a large part of the facial skeleton is derived from a newly identified, vertebrate-specific population of cells, called the cranial neural crest, that has its origins at the border of the dorsal neural plate (the future brain).Vertebrates develop from three germ layers—the endoderm, mesoderm, and ectoderm—which each give rise to distinct elements in the emerging body plan, and interactions between these layers are a common feature of embryogenesis. For example, early in development, cranial neural crest cells migrate to positions along the bottom  of the future head, where they form a series of developmental intermediate structures called pharyngeal arches. The arches facilitate interactions between crest cells (derived from ectoderm) and neighboring tissues (such as endoderm and surface ectoderm), which induce specific bone and cartilage patterns in the face. Recent chick studies showed that head endoderm, which contributes to the lining of the pharynx and gills, can pattern the facial skeleton. But the question remained, by what mechanism does endodermal signaling induce specific patterns of cartilage and bone?PLoS Biology, Justin Crump, Mary Swartz, and Charles Kimmel study the patterning of a jaw-support cartilage called the hyosymplectic in the larval zebrafish and find a “hierarchy of tissue interactions” at work. In zebrafish mutated for a gene called integrinα5, the authors report, a specific region of the hyosymplectic cartilage fails to develop. The loss of this cartilage region correlates with the loss of the first endodermal pouch. Pouches are outpocketings of the head endoderm that fuse with the skin to form the gill slits later in development. By labeling individual crest cells with fluorescent dye and making time-lapse recordings of these cells in transgenic fish, Crump et al. show that the hyosymplectic cartilage regions lost in the integrinα5 mutant are normally derived from crest cells directly adjacent to the first pouch.In this issue of Integrins are transmembrane receptors that promote cell adhesion and signaling. Although integrins function in crest cell migration, Crump et al. show that the Integrinα5 receptor is required in endoderm for hyosymplectic cartilage development and appears to promote development of the first pouch. The first pouch in turn acts as a template, by promoting both the survival and local clustering of crest cells, to pattern a specific region of the hyosymplectic cartilage.integrinα5 mutants also have region-specific defects in cranial muscles and nerves, the first pouch may serve to organize an entire functional unit in a region of the head. As the hyosymplectic element has undergone considerable change during evolution—from a jaw-support element in fish to a tiny, sound-conducting bone called the stapes in mammals—Crump et al. speculate that such a local, interconnected strategy of development would facilitate evolution of the vertebrate head. Changes in endodermal signaling would allow a particular skeletal element to vary in shape or size, in coordination with the muscles and nerves that move the skeletal element and independent of other regions of the head.But the pouch may have more far-reaching effects. Since It will be interesting to determine, the authors note, whether this hierarchical organization applies to other skeletal elements in the head. But for now, these results will inform efforts to understand the specificity of interrelated defects seen in human craniofacial syndromes such as DiGeorge Syndrome, whose underlying causes lie in the development of the endoderm.


Many trials of nebulized therapy have used nebulized saline as a "placebo". However, nebulized isotonic saline is sometimes used to assist sputum expectoration and relieve breathlessness in COPD patients. We designed this study to establish if nebulized saline had a placebo effect or a clinical effect.40 patients were studied following hospital admission for exacerbated COPD (mean FEV1 30% predicted). Patients were randomised to single-blind administration of either 4 mls of nebulized isotonic saline using an efficient nebulizer (active group n = 20) or an inefficient nebulizer (placebo group n = 20). Spirometry and subjective breathlessness scores  were measured before nebulized treatment and 10 minutes after treatment.There was no significant change in FEV1 after active or placebo nebulized saline treatment. Patients reported a 4% improvement in mean breathlessness score following placebo  compared with 23% improvement following active nebulized saline (p = 0.0001). 65% of patients given active nebulized saline but only 5% of the placebo group reported that mucus expectoration was easier after the treatment.This study lends support to the current use of nebulized saline to relieve breathlessness (possibly by facilitating sputum clearance) in COPD patients. Lung function was not affected. Nebulized saline can therefore be used as a placebo in bronchodilator studies involving COPD patients but it cannot be used as a placebo in trials assessing symptom relief. Nebulized saline is used by some doctors and physiotherapists to assist mucus clearance and to relieve breathlessness in patients with COPD, bronchiectasis and Cystic Fibrosis. This practice is justified by a small number of studies which have demonstrated enhanced sputum expectoration or improved breathlessness after nebulized saline or humidified oxygen -4. NebulHowever, nebulized saline has also been used as a placebo in several trials involving nebulized bronchodilator therapy ,11. For 40 patients were studied during a hospital admission for an exacerbation of COPD. Patients were recruited at a time when their condition had stabilized prior to their planned discharge from hospital. Clinical details of the patients are summarised in table Patients were randomised to receive 4 mls of 0.9% saline using an efficient nebulizer system (active group) or an inefficient nebulizer system (placebo group). The active nebulizer was a System 22 Acorn nebulizer (Medic-Aid, Bognor Regis UK Ltd) driven by the hospital's piped oxygen supply at a flow rate of 9 l/min for 10 minutes. This nebulizer system was found to deliver 95% of particles in the size range 2.5 to 2.8 microns using a Malvern laser system. . This small particle size was selected to achieve effective delivery to the airways. The placebo nebulizer was an old model (1980s) Bard Inspiron nebulizer (no longer manufactured) driven by oxygen at a flow rate of 3 l/min. This nebulizer system delivered 95% of particles in the size range 9.5 to 9.9 microns. This particle size was selected to achieve a placebo effect with deposition in the tubing of the system and in the pharynx but little penetration to the airways . Both neThe trial was conducted in a single-blind manner. 40 slips of paper were labelled "Treatment A" or "Treatment B" and placed in opaque brown envelopes. These were shuffled in random order and each patient was asked to select one envelope. This was then opened by the investigator and the appropriate treatment was administered (A active, B Placebo). For the six patients who took part in the study twice, the second treatment consisted of whichever treatment they had not received previously.Patients were told that we wished "to observe the effects of a nebulized treatment which is not a new or experimental drug". They were not informed of the exact nature of the nebulized treatments as this might have led patients to try to guess if the treatment which they received was a "placebo". The Ethics Committee agreed that it would not have been possible to measure a true placebo effect if patients were made aware that both treatments were saline (not a bronchodilator) and one of the nebulizers was deliberately made to run inefficiently.Patients were recruited on the Respiratory Wards of a University hospital. We recruited patients who had diagnosis of COPD confirmed by a respiratory consultant (patients with asthma or bronchiectasis were excluded from the study). Patients were approached by one of the investigators whilst in a relatively stable phase prior to discharge from hospital following an admission for exacerbated COPD. All testing was undertaken between 12.00 and 16.00, at least four hours after bronchodilator treatment.Prior to participating in the study, patients gave informed consent and undertook baseline measurement of FEV1 and FVC using the best of 3 blows on a Microlab 3300 Spirometer  was measured using a Wright's Peak Flow meter. Each patient also recorded an assessment of their perceived level of breathlessness using a seven point modified Likert scale (1 = Not breathless, 2 = Very mild breathlessness, 3 = Mild breathlessness, 4 = Moderate breathlessness, 5 = Severe breathlessness, 6 = Very severe breathlessness, 7 = Worst possible breathlessness).Ten minutes after completion of nebulized therapy, FEV1, FVC and PEF measurements and subjective breathlessness scores were repeated. Patients also recorded a subjective assessment of benefit using the following modified Likert scale. (1 = No benefit from this treatment, 2 = Very slight benefit, 3 = Slight benefit, 4 = Moderate benefit, 5 = Good benefit,, 6 = Very good benefit, 7 = Best possible benefit).Patients then received 4 puffs of salbutamol (400 mcg) using a Metered Dose Inhaler and 750 ml Volumatic spacer (Glaxo Smith Kleine UK).Fifteen minutes later, FEV1, FVC and PFR measurements and subjective breathlessness scores and symptom relief scores were repeated.All data was entered on a SPSS version 9 statistical package. Mann Whitney U-test was used to compare lung function tests and symptom relief scores. Wilcoxon Signed Rank test was used to compare the change in breathlessness scores for matched pairs before and after nebulized saline.The study was approved by Salford and Trafford Research Ethics Committee. All patients gave written informed consent to partake in the study and to receive a single dose of nebulized treatment .34 patients completed the study; patient details are summarised in table The baseline FEV1 of the two treatment groups was well matched. Both groups had a non-significant fall in FEV1 after nebulized saline therapy and a small rise in FEV1 after 400 mcg salbutamol from MDI-spacer Table . FVC, anThe placebo group had a 4% improvement in breathlessness after treatment (Wilcoxon p = 0.37) compared with a 23% improvement after active nebulized saline (Wilcoxon p = 0.0001). This corresponded to a reduction from 4/7 (moderate breathlessness) before treatment to 3/7 (mild breathlessness) after treatment in the active treatment group.The mean symptom relief score (patient's assessment of benefit) for the active treatment was 3/7, (slight benefit) almost identical to the response to 400 mcg salbutamol from MDI. The placebo group had a score of 2/7(very slight benefit) after nebulized placebo and 3/7(Slight benefit) after 400 mcg salbutamol from MDI.15 patients in the active group felt better after nebulized saline, 5 felt the same and no patient felt worse. Six patients in the placebo group felt better after nebulized treatment, 12 felt the same and 2 felt worse (Chi Squared test p = 0.013).Patients were asked if the nebulized treatments had any effect other than relief of breathlessness. 13 patients in the active group (65%) said that the nebulized treatment assisted sputum expectoration. Only 1 patient in the placebo group reported this effect (Difference between groups: -Fisher exact test, p = 0.0001). No patient reported any adverse effects from either nebulized treatment.This is the first study which has compared active nebulized saline with placebo nebulized saline. The results suggest that nebulized saline has non-bronchodilator therapeutic effects that are possibly explained by airway-moistening and sputum-inducing effects of nebulized saline. Sputum volume was not measured in the present study but two thirds of patients who were given nebulized saline through an efficient nebulizer system reported that it helped them to expectorate sputum. This finding is consistent with the results of previous studies which have shown improved sputum clearance and decreased breathlessness following the open administration of nebulized saline ,3 The rePrevious studies have shown that nebulized saline can have a bronchoconstrictor effect in some patients which is greater with hypertonic saline than with isotonic saline and greater in asthma patients than COPD patients ,6,8,9 NeThe main strength of the present study is inclusion of a placebo limb using an inefficient nebulizer system. Patients in the placebo group believed that they were receiving a nebulized treatment because a placebo effect could have been abolished if patients were told that both treatments involved no active medication and one of the patients involved an inefficient nebulizer system. This issue was discussed fully with the ethics committee and found to be acceptable because the patients did not miss any of their regular medication and they did not receive any pharmacological treatment. The use of a mouthpiece ensured that patients could not see or feel that the output from the experimental system was different to the nebulized bronchodilator therapy which they had received during their hospital admission . Furthermore, only 6 patients took part in both limbs of the study so most patients could not have tried to guess which treatment was more effective.The 23% improvement in breathlessness in the active group was equivalent to the subjective benefit following 400 mcg of salbutamol from MDI-spacer. This improvement in breathlessness occurred without bronchodilation, mirroring the findings of Poole et al . PatientThis study is in agreement with previous studies which have shown no bronchodilator effect  when nebulized saline is given to patients with COPD . This juHowever, as nebulized saline has non-bronchodilator therapeutic effects, it cannot be used as an inert placebo treatment in clinical studies where breathlessness or quality of life are to be measured. For example, Jenkins et al concluded that nebulized treatment had a strong placebo effect because patients expressed a preference for nebulized treatment even though the same bronchodilator effect could be achieved for most of their patients when nebulized saline was given with active bronchodilator therapy from a MDI device . It is lBritish and European nebulizer guidelines state that most patients with airflow obstruction should be treated with hand-held devices unless they have demonstrated clear additional benefit from the use of nebulized treatment in carefully monitored domiciliary studies ,14. The FEV1 Forced Expiratory Volume in 1 secondFVC Forced Vital CapacityPEF Peak Expiratory FlowCOPD Chronic Obstructive Pulmonary DiseaseMDI Metered dose inhalerThe authors declare that they have no competing interests.BROD developed the concept for the study and both authors designed the study protocol.SYK recruited patients and performed all study measurements.Both authors assisted in analysis of the data and preparation of the manuscript.The pre-publication history for this paper can be accessed here:


Swedish patient associations for breast cancer patients (PABCPs) offer patients with breast cancer unlimited meetings with a breast cancer survivor, a contact person (CP). We applied the voluntary action perspective in this interview study with members of Swedish PABCPs in order to explore how women with breast cancer experienced their contact with a CP from a PABCP.Audio-taped narratives from 8 women were analysed using Reissman's monitoring and Gee's analysis structure.Three themes appeared: 1. Shared experiences give new perspectives on having cancer, 2. Feelings of isolation are a part of the identity of the illness and 3. Relations with others enable self-help. However, the relationship with the CP is sensitive to timing, correct information and understanding.CPs act as sounding boards and should optimally have capacity for listening, gives support and act as partner in this conversation. On the other hand, CPs should be aware that their presence and limited general medical knowledge could at times disturb the patient's psychological recovery and strengthen feelings of isolation. Thus, PABCPs must be careful in selecting CPs and offer relevant educational activities related to the themes identified herein. Reach to Recovery Program (RtoRP), a world-wide rehabilitation program initiated in the U.S. in 1952 and was later established also in Europe. RtoRP is based on the opportunity to meet with individuals with similar experiences and thereby learn different ways of dealing with disease-related feelings and problems [Proximity to individuals and society in order to reach out and provide optimal support is basic for patient associations for cancer patients (PACPs) ,2. Swediproblems ,9.Rehabilitation from breast cancer is a requirement for becoming a CP in a PABCP according to the national organisation for Swedish breast cancer associations, (BRO) ,10. ListTreatment and voluntary action provide different perspectives in studies of self-help groups or mutual support groups in the voluntary sector. We have, in accordance with other researchers' views, chosen to apply the voluntary action perspective in this study of CPs activities within the voluntary sector -16. SuppWomen with breast cancer who had personal experience from CP activities were informants in this study. To qualify for participation, the informants were required not to be involved in treatment (surgery, chemotherapy, or radiotherapy) of the primary tumour. The cancer diagnosis should be within 3 years of the data collection in order to recall the illness and treatments obtained.We approached volunteers responsible for contact and visiting activities in 3 PABCPs who in their turn contacted 8 women who were judged eligible for the study. These women were given information on the purpose of the study and the interview procedure, and confirmation of confidential treatment of the women's information was sent by the PACP's. The recipients of the letter were informed that it was sent by the PABCP, but they were offered to contact the researcher with questions. The informants were asked to give their decision to the author (CC) by telephone within two weeks and all 8 individuals invited chose to participate in the study.The 8 informants had a mean age of 56 (range 39–69) years and demographic characteristics are presented in Table All the interviews were held in the home districts of the participants. The data were based on audio-taped narratives and takes into account people's natural way of constructing and interpreting experience and provides the opportunity to take relevant contextual factors into account . The perTranscription is a part of the analysis, regardless of whether the emphasis is on the content or the form . This stThe authors examined and discussed the stanzas and codas on the basis of their contents and relevance in relation to the aim. The final monitoring was done to find the informants' meaning according to their breast cancer when they met another individual with experience of breast cancer, i.e. a CP. The coda was used as the base in the final thematic monitoring  and we fEthical permission for the study was obtained from the Ethics committee at Lund University (LU 605-01).The thematic contents of the narratives' stanzas fell into the following themes, which were related to the purpose of the study:1. Shared experiences give new perspectives on having cancer2. Feelings of isolation are a part of the identity of the illness3. Relations with others enable self-helpThemes and sub-themes are presented in Table The meeting with the contact person can have emotional significance in terms of understanding that one is not alone in one's illness. Emotions came out in one case by a postcard sent by the CP. The following stanza from the informant exemplifies the CPs' activity.I got a card from my CPa (certain motif) where she wrote(name) I know what you're going throughI'm thinking about you (informant 2)The receiver kept the card and it gave her a warm feeling long after the most difficult time of the cancer illness was over.The contribution of the cancer association and the CPs to giving the women insight about their illness can take place through invitations to rehabilitation activities, such as physical therapy groups. One woman said that, in spite of her having many questions, she did not feel ready to have contact with the association other than coming to the therapy sessions, which gave that woman sufficient and obvious evidence that she was not alone. At the water therapy sessions, she was able to see that it was possible to survive cancer and said "it's nice to be able to, like feel that fellowship". The following stanza illustrates the activity.when we sit in the sauna – so everybody is looking -some have no breastsand some they've taken parts ofeverybody looks a little strange (informant 7)One woman said that her contact with her CP gave a long-lasting and negative experience of the meeting. After having received advice from a nurse, she had herself contacted the CP. Instead of getting access to other women's experiences through the CP she was left with an even stronger feeling of not only being ill but also of being odd. This feeling was caused by the CP not knowing that it was possible to have bilateral breast cancer at the same time (which this woman had). The CP had reacted with alarm – a reaction that frightened her and made her even more scared. These unpleasant feelings were still present after a year and because of the negative reactions she did not seek further contact with the CP, which led to a sense of embarrassment since she had initiated contact. The narrative illustrates the feelings isolation was strengthened by the CP and how the process of relating to the new disease identity was damaged.Another narrative came from a woman who felt odd because she did not identify herself as being ill. She felt that her cancer was less serious since she did not need further treatment after the operation. This feeling of being isolated gave her a great deal to think about, which she could talk about with her CP. The reaction of being ill came late, according to the informant, and was triggered when she wanted to donate blood. When she filled in the health certificate she realized she was not permitted to do so. The CP was available at that time to talk about the incident and the following two stanzas illustrate the importance that the CP was ascribed:it feels like she [the CP] understandswhat I mean, like she understandsthat I can float above itand thought like it wasn't so serious (informant 8)yes, I think thatshe took me seriouslywhen I felt like it was hardnot to be able to identify myself in the group [the PACP group] (informant 8)One woman described being one among others like oneself by talking about the special feeling that came over her when she was welcomed into the association. It was not necessary to say that she had had a breast cancer operation since everyone there knew; this was obvious in the association. Another woman described the importance of meeting people in the same age and situation in life, which took place at regular theme meetings for younger women. A younger woman exemplified the importance of being the same age by saying that women of the same age had common questions to discuss – questions that could not be talked about with health professionals. The following two stanzas illustrate how the women used the CP meetings to talk with women with similar problems:it could for example behow I could make up my eyes nowwhen I don't haveany eyelashes (informant 6)if there was anyonewho had childrenbecause the ones I'd met were only older peoplewho didn't have children living at home (informant 6)At each contact meeting, the women felt that they could gain knowledge through the different experiences that were described about different forms of treatment and their effects on well-being. One woman describes how she could balance her worry in this environment by talking about what she thought about, which is illustrated in the stanzas below:how will I go through this psychologicallyin the futurewill I trust this answeror how much will I worry (informant 2)alot of questions like thatthat I've thought aboutand that I couldtalk about with other people (informant 2)Another woman's reactions to some of the meetings were feelings of sadness, while other meetings could give her hope for the future. However, while the meetings made her react differently, she felt it was important to come to the CP meetings regularly to see with her own eyes that the other women were alive and led good lives. It came out in another narrative how it is gradually possible to talk with any of the members of the association that have experience even though it is the CP that is the person one initially talks with. Thus in the long run the experiences are felt as being most important, where the CP acts to open the door to them.Time, in the sense of a particular phase in the process, is important when the women want to make contact with others with experience of having breast cancer. One woman felt that she was not ready to contact the association and meet others with similar experience despite having many questions. At first, she said, she wanted most just to crawl into herself and manage on her own without involving other people. Nevertheless, this woman was content having received a card from the CP early in the process, telling her about the possibility of establishing a contact. The possibility of getting a contact is thus given higher priority than hearing others' experiences at an early stage.One woman evaluated the meeting with the CP positively in part because the CP was a trained CP and in part because the CP knew what she needed to hear. This is illustrated in the following stanza:it was most of all goodto hear [name of CP]maybe because she had trainingknew what you needed to hear (informant 8)Another woman described how she had used her CP as a "sounding board" in her choice of surgical method. It comes out in this narrative that the sounding board function and not the counselling function made the woman finally decide in favour of a surgical method that the CP had not recommended on the basis of her own experience. The fact that the CP had given an opinion helped the woman to form an opinion herself. Thus it is not necessary to share the exact same experience; at times it is enough to share the experience of cancer.One of the women said that she would have appreciated there being someone with experience to contact when she was forced to wait for a long period before having her operation. Access to a CP for one's own personal problems is illustrated in the following stanza:I know she's the contact personwhich makes me thinkthat I can turn to herand talk about my special problems (informant 8)Women describe not only the benefit of the CP meetings but also say that there can be a feeling of more or less indifference in the contact with the CP. In one example the CP acted as a guide to the association's premises but had otherwise not spoken of anything in particular. Another woman had come into contact with the association's CP at an educational class but had never contacted a CP herself. For these women it was sufficient to contact with other women in educational classes organised by professionals.Since the CP activity is central within the PABCPs, we aimed to investigate how women with breast cancer described their experiences from having contact with a CP. The narratives allowed the women to reflect and formulate themselves through this opportunity to tell their own stories with the aim to analyse their experiences . The strThe narratives in our study illustrate the importance of meeting women of the same age and in a similar life situation and of being in an environment (CP meeting) that is free of need to explain. Meeting others and experiencing their' reactions help these women feel normal . "CatchiEven though all patients do not wish to be confronted with the experiences of others via personal contact with a CP, they may be ready to be confronted with the disease in other ways. A confrontation with women who have undergone partial or complete mastectomies can take place in water therapy sessions and in the sauna. The results show that this meeting also gave a feeling of fellowship. This may mean that the person who chooses only this kind of activity is ready only for visual impressions. Breasts mean different things to different women, and only the individual woman can define how great a handicap breast surgery is. Langellier and Sullivan  studied It is also important that a CP is able to manage timing, in the sense of the phase in the process at which contact is made with others who have experience or when the person is ready to confront others' experiences. Great sensitivity towards individual needs is required and there are individual variations as to the optimal time for a meeting, which are related to when the individual is ready to encounter other people's experiences. In terms of being ready, thoughts arise as to the many visual and auditory impressions that cancer patients receive during a treatment session, e.g. in a waiting room and when given treatment, regardless of whether they are ready for these or not.It is conceivable that some individuals do not have any need to meet with other people and share their experiences since recovery can also occur without involving others. However, it is not only the initial need for contact that varies. Women also have different needs in terms of the duration of the contact. For one person, contact with a CP may simply mean guidance in the association's activities while for another a CP can act as a support person for a period of several years and the information-seeking behaviour can also change over time .For the woman who felt great isolation in the illness the meeting with the CP strengthened the feelings of isolation, which demonstrates how the meeting with the CP led to a non-intended consequence. This demonstrates the need for the PABCPs to be meticulous in the choice of CPs; the requirements must not only be that they have been rehabilitated but also that they have substantial knowledge about cancer, treatment possibilities, and psychological reactions to disease. Although self-help may come through a relation with other individuals with a shared experience  the assoThe results also demonstrate that some questions could not be answered by health-care professionals, but rather by individuals with personal experience from cancer. Hence, new knowledge developed in the meeting with the CP  declare that they have no competing interests.CC: study design, data collection, data analysis, and writing the manuscript.MN: supervising the study and participation in writing the manuscript.KN: study design, data analysis, supervising the study, and participation in writing the manuscript.The pre-publication history for this paper can be accessed here:


In this study we have used whole cell patch clamp recording and fura-2 Ca2+ imaging to characterise the actions of pregabalin on the electrophysiological properties of cultured dorsal root ganglion (DRG) neurones from neonatal rats. The aims of this study were to determine whether pregabalin and gabapentin had additive inhibitory effects on high voltage-activated Ca2+ channels, evaluate whether the actions of pregabalin were dependent on GABA receptors and characterise the actions of pregabalin on voltage-activated potassium currents.Gabapentin and pregabalin have wide-ranging therapeutic actions, and are structurally related to the inhibitory neurotransmitter GABA. Gabapentin, pregablin and GABA can all modulate voltage-activated Ca2+ current in cultured DRG neurones. The residual Ca2+ current recorded in the presence of pregabalin was sensitive to the L-type Ca2+ channel modulator, Bay K8644. Saturating concentrations of gabapentin failed to have additive effects when applied with pregabalin, indicating that these two compounds act on the same type(s) of voltage-activated Ca2+ channels but the majority of Ca2+ current was resistant to both drugs. The continual application of GABA, the GABAB receptor antagonist CGP52432, or intracellular photorelease of GTP-γ-S had no effect on pregabalin-induced inhibition of Ca2+ currents. Although clear inhibition of Ca2+ influx was produced by pregabalin in a population of small neurones, a significant population of larger neurones showed enhanced Ca2+ influx in response to pregabalin. The enhanced Ca2+ influx evoked by pregabalin was mimicked by partial block of K+ conductances with tetraethylammonium.Pregabalin (25 nM – 2.5 μM) inhibited 20–30% of the high voltage-activated Ca+ currents, the inhibitory effect of pregabalin was prevented with apamin. The delayed enhancement of K+ currents was attenuated by pertussis toxin and by intracellular application of a (Rp)-analogue of cAMP.Pregabalin produced biphasic effects on voltage-activated K2+ and K+ channels. The pharmacological activity of pregabalin is similar but not identical to that of gabapentin. The actions of pregabalin may involve both extracellular and intracellular drug target sites and modulation of a variety of neuronal conductances, by direct interactions, and through intracellular signalling involving protein kinase A.Pregabalin reduces excitatory properties of cultured DRG neurones by modulating voltage-activated Ca We had two specific aims for this project. The first was to determine whether gabapentin and pregabalin have the same mechanisms of action, by examining whether saturating concentrations of gabapentin and pregabalin act in an additive manner to attenuate Ca2+ influx. The second aim was to further evaluate GABA receptors as target sites for these drugs in sensory neurones. This latter element to the study was conducted because it has been proposed that specific GABAB receptors with a gb1a-gb2 heterodimer composition are the sites of agonist activity of gabapentin. These specific receptors can be coupled to an inwardly rectifying K+ channel and / or voltage-activated Ca2+ channels to dampen neuronal electrical excitability [B receptor agonist, baclofen, have different actions. Weaver mutant mice (wv/wv) are insensitive to both gabapentin and baclofen, however in control littermates, Weaver control mice (+/+, wv/+), only baclofen evoked a K+ current [In this present study the effects of pregabalin and gabapentin on the electrophysiological properties of cultured neonatal rat DRG neurones were measured with particular reference to Catability -15. Howe current .p < 0.01). Pregabalin (2.5 μM) reversibly reduced the number of action potentials during 300 ms depolarisations from 8 to 3 (n = 5, p < 0.01). However, pregabalin and gabapentin did not significantly alter the properties (amplitude, duration & threshold) of single action potentials evoked by 5 ms depolarising current commands.The firing of multiple action potentials in response to a sustained depolarising current command is a property of a sub-population (under 20 %) of cultured DRG neurones. Application for 3 minutes, of either pregabalin PGB; 2.5 μM) or gabapentin  reduced the frequency of action potential spikes evoked by 300 ms depolarising current step commands applied every 30 s [ μM or ga2+ currents (ICa) recorded from DRG neurones were reversibly attenuated  by 2.5 μM gabapentin. Similarly, 3 minutes application of pregabalin  but in 20 neurones from the same cultures pregabalin evoked a mean increase in Ca2+ flux to 185 ± 20 % (p<0.05) of the control. Raising the pregabalin concentration to 25 μM increased the proportion of inhibitory responses (n = 21 out of 49 neurones) but enhancement of Ca2+ transients was still observed in the remaining 28 neurones . Although in some cells good recovery from pregabalin actions was observed (figure +-evoked Ca2+ transients was only 8.4 ± 1.5 % (n = 39). Similarly, the mean control level of increase in K+-evoked Ca2+ transients was only 7.2 ± 1.3 % (n = 35).Given the variable and rather modest but reversible inhibitory actions of pregabalin on Iansients ,12. Pregd figure  the inhi+-evoked Ca2+ transients by pregabalin was mainly seen in neurones with larger cell somas and that in smaller neurones pregabalin produced inhibitory effects.To investigate the different responses to pregabalin, the sizes of cell somas were measured and compared. Although there is overlap, figure 2+-dependent conductances of DRG neurones are sensitive to ryanodine and Ca2+-induced Ca2+ release has been reported in these neurones. Modulation of either Ca2+-induced Ca2+ release and / or Ca2+ homeostatic mechanisms might provide a mechanism by which pregabalin enhanced Ca2+ transients in neurones with intermediate and large sized cell somas. This was investigated in two ways. Firstly, the actions of pregabalin on caffeine-evoked Ca2+ transients were evaluated in nominally Ca2+-free extracellular conditions (NaCl-based solution with no added CaCl2). Single caffeine (1 mM) responses were obtained from DRG neurones in either the absence or presence of 25 μM pregabalin. No differences in either amplitudes or durations of Ca2+ transients were seen when 8 control caffeine responses were compared with 4 caffeine responses obtained in the presence of pregabalin  was applied with NaCl-based extracellular medium and DRG neurones were stimulated with 30 mM KCl. Attenuation of potassium conductances by TEA markedly increased the K+-evoked Ca2+ flux in all eight DRG neurones studied, mimicking in part the action of pregabalin  and gabapentin (2.5 μM) produced modest but significant inhibition of ICa at the peak of the current and at the end of the stimulus , 2.5 μM pregabalin alone  and 2.5 μM gabapentin and 2.5 μM pregabalin applied together .Pregabalin and gabapentin have related chemical structures and appear to have similar but usually modest inhibitory effects on Cas Figure . However+ evoked Ca2+ flux and the effects of both ligands. Our previous experiments with gabapentin (25 μM) had only identified inhibitory effects using this experimental approach [+ evoked Ca2+ influx  of the control response to 30 mM K+. Consistent with the electrophysiological experiments, this level of inhibition was similar to the inhibitory effects of pregabalin and gabapentin applied separately.In spite of the added complexity of mixed responses with pregabalin, experiments were carried out using fura-2 to measure Kapproach ,12. Simux Figure  &6C. Pre2+ imaging data show that no additive inhibitory effects were found during simultaneous application of saturating concentrations of gabapentin and pregabalin. The data therefore support the contention that both drugs have a closely related or a common site of inhibitory action. The data also indicate that in the modulation of Ca2+ channels these drugs do not act in an additive manner even though a substantial proportion of current is resistant to both drugs. It should be emphasized however that pregabalin can reversibly enhance K+-evoked Ca2+ transients, an effect not seen with gabapentin. Therefore there may be other additional actions of pregabalin that can be identified in Ca2+ imaging experiments when all membrane conductances are intact.The electrophysiological and fura-2 CaB receptor antagonist, CGP52432 [2+ in 18 of 41 DRG neurones studied  in the proportion of neurones that were inhibited by pregabalin. It is not clear why this is but it may reflect the sensitivity of Ca2+ transients evoked in DRG neurones, which can only be evoked consistently in most DRG neurones by three depolarising stimuli. Thus the GABA-evoked responses may influence Ca2+ homeostatic mechanisms and subsequent stimulated Ca2+ entry. However, the critical observation from these experiments is that GABA receptor desensitisation does not prevent either Ca2+ transient enhancement or inhibitory actions of pregabalin in neurones that were sensitive or insensitive to GABA.In this section of the study two strategies were used to evaluate the possible roles of GABA receptors in pregabalin responses in cultured DRG neurones. Firstly, pregabalin actions were studied in the presence of a saturating concentration of GABA; this initially activated all types of GABA receptor and then caused desensitization of these receptors. Secondly, the effect of a potent and selective GABACGP52432  on pregad Figure  &7B. Thex Figure ; Table 2Ca taking place through activation of G-protein coupled GABAB receptors, was then assessed. CGP52432 (10 μM) when applied alone had no effect on ICa. In the presence of the GABAB receptor antagonist CGP52432 (10 μM), pregabalin (2.5 μM) evoked a 27 ± 3 % (n = 9) inhibition of the voltage-activated Ca2+ current and complete recovery was observed 5 minutes after removal of the drug-containing pipette  of control) in response to pregabalin and 12 neurones showed inhibition of Ca2+ influx (64 ± 8 % (n = 12) of control) in response to pregabalin . During this period there was a clear reduction in the inward current, although a steady state current level was reached. However, subsequent application of pregabalin (2.5 μM) resulted in a further significant reduction in Ca2+ current amplitude to -0.43 ± 0.07 nA (n = 7, p<0.05). This represents a mean inhibition of 22 ± 5 % by pregabalin, a value very similar to the percentage inhibition produced by pregabalin in the absence of SCH-202676 (21 ± 3%). Therefore SCH-202676 had no effect on the pregabalin responses, indicating that pregabalin was not acting through an SCH-202676-sensitive G-protein coupled receptor (data not shown). Preliminary Ca2+ imaging experiments were also conducted to determine whether extracellular SCH-202676  altered the effects of pregabalin (2.5 μM) on K+-evoked Ca2+ transients. Under these experimental conditions pregabalin was found to markedly enhance or inhibit K+-evoked Ca2+ transients (data not shown). This data again suggests that pregabalin was not acting through mechanisms that were sensitive to SCH-202676.SCH-202676 (10 μM) was applied to the intracellular environment via the patch pipette solution. After 5 minutes equilibration the whole cell Ca2+ current was obtained, three 200 V flashes of intense near UV light were applied to the neurone to achieve intracellular flash photolysis of the caged GTP-γ-S. We estimate that approximately 15 μM GTP-γ-S was photoreleased by the three flashes. As previously observed intracellular flash photolysis of caged GTP-γ-S reduced the amplitude and slowed the activation of ICa [2+ channels , when applied after photorelease of GTP-γ-S, pregabalin produced a 20 ± 10 % (n = 5) inhibition of ICa.Caged GTP-γ-S (100 μM) was applied to the intracellular environment via the patch pipette solution and after entering the whole cell recording configuration DRG neurones were left for 5 minutes to equilibrate. Once a stable control Can of ICa . The effn of ICa , which is Figure . No recod Figure . In anotd Figure . When pr2+ imaging experiments that not all the cellular actions of pregabalin could be explained by the inhibition of voltage-activated Ca2+ channels because both inhibition and enhancement in K+ evoked Ca2+ flux was observed. To investigate the actions of pregabalin further, its' effects on voltage-activated K+ currents in DRG neurones were studied. Previously, Stefani and colleagues found that gabapentin modulated neuronal steady state non-inactivating K+ currents [It was clear from the Cacurrents .+ current activated from a holding potential of -90 mV by a 100 ms voltage step command to 0 mV  applied for 3 to 5 minutes produced an enhanced K+ current in 11 out of 21 neurones but a modest inhibition of the outward current in 10 out of 21 neurones  had no significant effect on the voltage-activated KV Figure . Howevers Figure  and 10C. neurone . To dist+ currents in DRG neurones  15 minutes after perfusion of 250 μM pregabalin. Figures + currents were recorded from DRG neurones for 16 minutes (n = 10).Long-term modulation of K+ current modulation. Apamin, a toxin from the honeybee, was used to block small conductance Ca2+-activated K+ currents. Apamin (1 μM) caused a reduction in outward current at +40 mV from 2.64 ± 0.33 nA to 2.37 ± 0.39 nA, subsequent application of 250 μM pregabalin enhanced the current to 3.02 ± 0.39 nA . No inhibitory effects were seen with pregabalin after treatment with apamin, suggesting that it is the apamin-sensitive Ca2+-activated K+ channels that are inhibited by pregabalin  evoked an increase in the K+ current that started to develop within the first minute of entering the whole cell recording configuration. After an initial increase that could reflect equilibration with the KCl-based patch pipette solution containing pregabalin, a slow sustained significant increase in the outward K+ current continued to develop over a 15 minute period  prevented pregabalin-induced long-term K+ current enhancement in cultured DRG neurones -cAMP [+ current (n = 9). This may indicate that PKA has little or no basal or tonic activity on K+ currents in DRG neurones in culture. However, when pregabalin was applied to DRG neurones loaded with (Rp)-cAMP no long-term enhancement of the outward current was observed. Under these recording conditions pregabalin still produced some inhibition of the K+ current  in the pregabalin-induced enhancement of KRp)-cAMP . Intracet Figure  &14D. Th2+ channels and that these inhibitory actions are independent of GABA receptor activation. Some features of the actions of pregabalin on intermediate size and large DRG neurones appear not to be seen with gabapentin. However, these distinct responses involve enhanced K+-evoked Ca2+ transients by pregabalin rather than inhibition of Ca2+ channels.In conclusion, these results indicate that pregabalin acts via the same basic mechanisms as gabapentin to inhibit voltage-activated Ca2δ subunits of voltage-activated Ca2+ channels remain a possible site of action for both pregabalin and gabapentin. Dooley and colleagues showed that both gabapentin and pregabalin attenuated K+-evoked norepinephrine release from rat neocortical slices by inhibiting P/Q-type Ca2+ channels [2+ channels [B receptors, gabapentin selectively inhibited N-type Ca2+ channels in hippocampal pyramidal neurones [2+ channel modulation seen with the GABAB receptor agonist, baclofen. Our previous investigation using "toxityping" showed that gabapentin inhibited a variety of Ca2+ channels in DRG neurones [2δ Ca2+ channel subunits. These α2δ subunits are not specifically combined with distinct pore forming Ca2+ channel subunits (α1) in all neurones and may therefore inhibit pharmacologically diverse Ca2+ channels depending on the expression of Ca2+ channel subunits in different neurones. However, transfection studies have shown that oocytes expressing Ca2+ channels (Cav2.2) containing β1b and α2δ-1 or α2δ-2 subunits are insensitive to acute application of 50 μM gabapentin [2δ – Ca2+ channel subunits are involved in the modulation of neuronal excitability by gabapentin. Additionally, the modulation of voltage-activated Ca2+ channels by gabapentin and pregabalin acting through indirect mechanisms has been suggested. Candidates for such indirect mechanisms include the control of Ca2+ channel functional expression [Alphachannels . In cortchannels . Our worchannels  and pregneurones . Interesneurones . We concbapentin . This wopression ,30 and mpression .+ and Ca2+ channels by gabapentin [2+ channels also appears unlikely in DRG neurones. This is because in this study we found that intracellular flash photolysis of GTP-γ-S had no influence on pregabalin-evoked current inhibition and pregabalin did not alter responses to GTP-γ-S. Our findings are in agreement with those made in a previous study on cells expressing GABAB1a/B2 or GABAB1b/B2 receptor subunits. In this study GABA and the GABAB receptor agonist baclofen evoked [35S]-GTP-γ-S binding responses but even at high concentrations both gabapentin and pregabalin did not [In hippocampal pyramidal neurones, uncoupling G-proteins from metabotropic receptors with N-ethylmaleimide prevents modulation of Kbapentin . However did not .B receptors did not attenuate pregabalin actions. These findings add to the published studies that indicate that gabapentin and pregabalin effects are independent of GABA receptor activation at least in some preparations [- conductance to alter membrane potential and input resistance and the GABAB receptor antagonist saclofen did not influence the inhibitory action of gabapentin on ICa [The experiments designed to assess potential roles of GABA receptors in the responses to pregabalin showed that GABA receptor desensitization or the blockade of GABAarations ,32. Specn on ICa .2δ subunits than gabapentin and in a number of studies is more effective. When applied alone pregabalin produced enhancement in Ca2+ flux in some neurones, an effect not seen with gabapentin. So, it was surprising to see in the imaging experiments that when gabapentin and pregabalin were applied together they produced only inhibitory effects. This may reflect the allosteric interactions between these drugs and α2δ subunits of Ca2+ channels as well as indirect modulation of Ca2+ dependent conductances and interactions with components of cell signalling.Pregabalin has a higher affinity for α2+ currents are compared with effects on K+-evoked Ca2+ transients. This may in part be due to effects of pregabalin on membrane conductances in addition to voltage-activated Ca2+ currents. These effects may not be detected under voltage clamp recording conditions where Na+ and K+ currents are blocked to isolate Ca2+ currents. In the imaging experiments all voltage-activated conductances are intact and may be modulated by pregabalin. Alternatively, pregabalin could potentially have a direct or indirect influence on Ca2+-induced Ca2+ release from intracellular stores and alter Ca2+ homeostatic mechanisms. These effects may be modulated differently in different sub-populations of DRG neurones and so produce the mixed responses recorded in small, intermediate and large neurones in this study. Thus different actions of pregabalin may be detected using fura-2 fluorescence imaging but these additional mechanisms may not influence the measurements of Ca2+ currents. Although mixed responses to gabapentin were not previously identified, neither were clear effects on the amplitude of K+-evoked Ca2+ transients apparent in DRG neurones [2+-induced Ca2+ release or Ca2+ homeostatic mechanisms such as Na+/Ca2+ exchange. This conclusion was reached because pregabalin did not influence Ca2+ transients evoked by caffeine and still enhanced K+-evoked Ca2+ transients in choline chloride-based (low Na+) extracellular solution.There appear to be some inconsistencies in the pregabalin data when its actions on voltage-activated Caneurones , althougneurones . These a2+ imaging experiments prompted us to investigate the actions of pregabalin on voltage-activated K+ currents, which could be modulated to cause an increase in Ca2+ influx. This speculation was supported by the finding that application of the K+ channel inhibitor, TEA, enhanced K+-evoked Ca2+ transients. Stefani and colleagues have reported inhibition of outward K+ currents by gabapentin [+-evoked [3H]-noradrenaline release. The activation of KATP channels is implicated in these responses because not only does glibenclamide, a KATP channel antagonist, attenuate the gabapentin response but also pinacidil, a KATP channel agonist, mimics the response and did not have additive effects with gabapentin [The mixed responses to pregabalin seen in the Cabapentin . Additiobapentin .+ currents observed. The inhibitory actions of both gabapentin and pregabalin on K+ currents recorded from DRG neurones may not reflect a direct action of these drugs on K+ channels. The effects of apamin, which prevented the inhibition of outward current by pregabalin indicated that small conductance Ca2+-activated K+ channels were involved in the response. This is consistent with the effects of gabapentin seen in isolated cortical neurones [2+ channels may underlie the inhibition of outward K+ current. A reduction in Ca2+ influx would lead to reduced activation of Ca2+-activated K+ channels and therefore a smaller outward current. If this indirect effect of pregabalin resulted in a disproportionate reduction in Ca2+-activated K+ conductances this may result in prolonged depolarisation. In turn a prolonged depolarisation may maintain activation of Ca2+ channels that are insensitive to pregabalin and an increase in Ca2+ influx as seen in a sub-population of larger DRG neurones in the Ca2+ imaging experiments. It is not clear why gabapentin, which produced modest inhibition of K+ currents, did not also produce enhancement of K+-evoked Ca2+ transients. It may be explained by a balance between inhibition of Ca2+ influx through voltage-activated channels and modulation of Ca2+ flux through inhibition of K+ conductance. Consistent with this hypothesis, gabapentin appears to be less effective than pregabalin at modulating K+-evoked Ca2+ transients in DRG neurones. This is supported by our previous study that showed that gabapentin did not significantly alter the peak Ca2+ transients [Mixed or biphasic responses to gabapentin and pregabalin were seen, with both inhibition and enhancement of Kneurones . The preansients .+ currents by gabapentin and pregabalin is a powerful mechanism for reducing cell excitability. The slow development of this response and its gradual decline suggested that intracellular signalling events were involved. Pregabalin produces enhancement of K+ currents when applied either inside or outside DRG neurones. The delay in the development of these responses is less with intracellular application of pregabalin, suggesting that it may have an intracellular site of action. This effect may also depend on drug uptake into cells via L α-amino acid transporters and the presence and activity of these transporters may greatly influence cell sensitivity to gabapentin and pregabalin. Although it is not clear how gabapentin and pregabalin might activate PKA, it is a candidate target site. Our previous work showed that inhibition of Ca2+ currents by gabapentin was sensitive to cAMP analogues that activated or inhibited PKA [+ currents by pregabalin. In the literature there are a number of reports of PKA altering neuronal excitability and modulating K+ conductances. PKA is involved in pain signalling, playing a role in prostaglandin-induced activation and sensitization of DRG neurones [+ conductances by prostaglandin E-2 [+ channel activity [+ and Ca2+ channels by a synthetic cannabinoid receptor agonist and the sensitivity of these responses to Wiptide, a PKA inhibitor [The delayed enhancement of voltage-activated Kited PKA . Similarneurones . PKA actneurones  and A-tyneurones  and is indin E-2 . Our resndin E-2 , large cactivity  and cannactivity . In the nhibitor . Howevernhibitor  could de2+ and K+ conductances may underlie this characteristic.An interesting feature of some actions of gabapentin and pregabalin is that their effects often outlast the period of drug delivery. Maintained intracellular signalling effects of these drugs and the delayed responses involving PKA modulation of Ca2+ channels with resulting reduced activation of Ca2+-activated K+ currents and effects on voltage-dependent ion channel availability. Although the delayed enhancement of voltage-activated K+ channels could contribute, this effect appears to develop over a longer period. These actions may contribute both to anticonvulsant effects of gabapentin and pregabalin as well as their therapeutic actions in pain disorders. It is particularly worth noting that the inhibition of Ca2+ influx is most consistently seen in small diameter cultured DRG neurones that are likely to be the pain fibres.Gabapentin has previously been shown to attenuate high frequency action potential firing both in CNS neurones  and in c+-evoked neurotransmitter release in CNS neurones [2+ channels through drug interactions with α2δ subunits, reduced activation of Ca2+-dependent conductances and modulation of both Ca2+ and K+ channels through metabotropic mechanisms that involve PKA. Interestingly, both gabapentin and pregabalin inhibit release of sensory peptides (substance P and CGRP) this may provide a mechanism of action down-stream of ion channel modulation. This effect is only observed after inflammation and may contribute to their analgesic properties of gabapentin and pregabalin [Our data adds to previous work that has assessed the actions of gabapentin and pregabalin on Kneurones  and the neurones ,9,42-44.egabalin .2+ channels in DRG neurones are targets for antinociceptive agents but the pharmacology of these channels remains to be fully exploited in pain therapy [Ca therapy ,47. Furt3 (14 mM), streptomycin (50 μg/ml) and penicillin (50 IU/ml). The cultures were maintained for up to two weeks at 37°C in humidified air with 5% CO2. Cultures were re-fed with fresh media after 5 days.One to four-day old Sprague-Dawley rats were decapitated and dorsal root ganglia removed. DRG neurones were dissociated enzymatically (0.125% collagenase for 13 minutes and 0.25% trypsin for 6 minutes) and mechanically (trituration). Primary cultures of DRG neurones were plated on lamin-polyornithine coated coverslips and bathed in Ham's F-14 culture medium  containing 10% horse serum (Gibco), low NGF , NaHCO2+ channels and other potential effectors [In some experiments DRG neurones were pre-treated with pertussis toxin  to ADP-ribosylate the α subunits of certain G-proteins. This prevents pertussis toxin-sensitive G-protein being activated through a range of G-protein coupled receptors and inhibits coupling to voltage-activated Caffectors ,48.2+ imaging were used to measure the inhibitory actions of pregabalin and gabapentin on Ca2+ entry through voltage-activated channels. Initially, multiple firing properties of a sub-population of DRG neurones were studied using a patch pipette solution containing in mM: KCl, 140; EGTA, 5; CaCl2, 0.1; MgCl2, 2.0; HEPES, 10.0; ATP, 2.0. The extracellular solution containing in mM: NaCl, 130; KCl, 3.0; CaCl2 2.0; MgCl2, 0.6; NaHCO3 1.0, HEPES 10.0 and glucose 5.0. For recording Ca2+ channel currents the patch pipettes were filled with CsCl-based solution containing in mM: 140 CsCl, 0.1 CaCl2, 5 EGTA, 2 MgCl2, 2 ATP, 10 Hepes. The pH and osmolarity of the patch pipette solutions were corrected to 7.2 and 310–320 mOsm.l-1 with Tris and sucrose. The extracellular bathing solution used to study Ca2+ currents contained in mM: 130 choline chloride, 2 CaCl2, 3 KCl, 0.6 MgCl2, 1 NaHCO3, 10 HEPES, 5 glucose, 25 tetrethylammonium chloride, 0.0025 tetrodotoxin (Sigma). The pH and osmolarity of this extracellular bathing solution was corrected to 7.4 and 320 mOsml-1 with NaOH and sucrose respectively. The recording solutions used in these experiments were designed to attenuate voltage-activated Na+ and K+ currents and isolate voltage-activated Ca2+ currents. The range of values for the series resistance under our recording conditions was from ~8 to 15 MΩ. Pregabalin and other drugs were applied to the extracellular environment by low-pressure ejection from a blunt pipette positioned about 50–100 μm away from the cell being recorded. Voltage-activated Ca2+ currents were evoked by 100 ms voltage step commands applied every 30 s.The whole cell patch clamp recording method and fura-2 Ca+ currents but NaCl-based extracellular bathing solution and KCl-based patch pipette solutions were used.Similar protocols were used to study the actions of pregabalin on voltage-activated K2+ currents intracellular flash photolysis was achieved by flashing the neurone being studied (three 200 V flashes to produce ~15μM GTP-γ-S) using a XF-10 xenon flash lamp with a UG11 bandpass filter  was included in the CsCl-based patch pipette solution. After obtaining control Caentific; )2+ and K+ currents had scaled linear leakage and capacitance currents subtracted to obtain values for the net inward Ca2+ current or net outward K+ current. Data are given as mean ± standard error of the mean (s.e.m.) values and statistical significance was determined using a paired or independent Student's t test as appropriate.All voltage-activated Ca2+ imaging in cultured DRG neurones the cultures were incubated for 1 hour in NaCl-based extracellular solution contained (in mM): NaCl, 130; KCl, 3.0; MgCl2, 0.6; CaCl2, 2.0; NaHCO3, 1.0; HEPES, 10.0; glucose, 5.0 and fura-2AM, 0.01; (Sigma, 1 mM stock in dimethylformamide). The pH was adjusted with NaOH to 7.4 and the osmolarity to 310–320 mOsm with sucrose. The cells were then washed for 10–20 minutes with NaCl-based extracellular solution to remove the extracellular fura-2AM and this period allowed cytoplasmic de-esterification of the Ca2+ sensitive fluorescent dye. The cells were constantly perfused with NaCl-based extracellular solution (1–2 ml/min) and viewed under an inverted Olympus BX50WI microscope with a KAI-1001 S/N 5B7890-4201 Olympus camera attached. Some Ca2+ imaging experiments were carried out using a Ca2+-free NaCl-based solution (as standard NaCl-based extracellular solution but with no added CaCl2) or choline chloride-based solution (as standard NaCl-based extracellular solution but with choline chloride in place of NaCl). The fluorescence ratiometric images from data obtained at excitation wavelengths of 340 nm and 380 nm were viewed and analysed using OraCal pro, Merlin morphometry temporal mode (Life Sciences resources, version 1.20). The DRG neurones were stimulated with NaCl-based extracellular solution containing high K+ (30 mM), which produced depolarisation, activation of voltage-gated Ca2+ channels and large transient increases in intracellular Ca2+. Three consistent transient increases in intracellular Ca2+ could be obtained in a single experiment on cultured DRG neurones [2+ transient amplitude, duration at 1/2 peak amplitude and total Ca2+ flux were measured. All experiments were conducted at room temperature and data are expressed as means ± s.e.m.For Caneurones . The actDRG, Dorsal root ganglion.cAMP, Cyclic adenosine monophosphateGABA, Gamma aminobutyric acidGBP, GabapentinGTP-γ-S, Guanosine 5'-o(3-thio)triphosphateCa, Calcium currentINGF, Nerve growth factorPGB, PregabalinPKA, Protein kinase APP, Pre-pulse(Rp)-cAMP, (R)-adenosine, cyclic 3', 5'-(hydrogenphosphorothioate) triethylammonium2+ imaging experiments. DM and RHS designed and conducted the experiments on K+ currents and wrote the first draft of this manuscript.All the authors of this manuscript contributed to electrophysiological and Ca


However, clinically useful alternative approaches for introducing p53 activity into cancer cells are clearly needed. It has been hypothesized that direct reactivation of endogenous p53 protein in cancer cells will be therapeutically beneficial, but few tests of this hypothesis have been carried out in vivo. We report that a transducible D-isomer RI-TATp53C′ peptide activates the p53 protein in cancer cells, but not normal cells. RI-TATp53C′ peptide treatment of preclinical terminal peritoneal carcinomatosis and peritoneal lymphoma models results in significant increases in lifespan (greater than 6-fold) and the generation of disease-free animals. These proof-of-concept observations show that specific activation of endogenous p53 activity by a macromolecular agent is therapeutically effective in preclinical models of terminal human malignancy. Our results suggest that TAT-mediated transduction may be a useful strategy for the therapeutic delivery of large tumor suppressor molecules to malignant cells in vivo.Advanced-stage peritoneal carcinomatosis is resistant to current chemotherapy treatment and, in the case of metastatic ovarian cancer, results in a devastating 15%–20% survival rate. Therapeutics that restore genes inactivated during oncogenesis are predicted to be more potent and specific than current therapies. Experiments with viral vectors have demonstrated the theoretical utility of expressing the  Specific activiation of the tumor suppressor protein p53, using a transducible p53 C-terminal peptide, dramatically increases survival in a mouse model of peritoneal carcinomatosis. This peptide offers therapeutic potential for tumors in which p53 is mutated Most patients who succumb to cancer do so not from primary tumor burden, but from metastatic disease . For exaA significant effort has been aimed at understanding the function of tumor suppressor gene pathways that are genetically and epigenetically altered during oncogenesis . One ratp53, in cancer cells , are capable of traversing the plasma membrane and entering the cytoplasm of cells by a concentration-dependent, but receptor-independent, macropinocytic mechanism . PTDs hap53 structural mutations. In contrast, primary cells are resistant to p53C′ peptide action  is a therapeutically effective means of activating the p53 tumor suppressor pathway in preclinical models of terminal metastatic cancer.Although PTDs solve one major obstacle to the use of intracellular peptides as therapeutics, the susceptibility of peptides to degradation in vivo remains problematic. To circumvent the problem of proteolytic degradation, we synthesized a retro-inverso version of the parental p53C′ peptide by inverting the peptide sequence, using D-isomer residues, and adding the TAT PTD to obtain a transducible RI-TATp53C′ peptide A. This dTo determine whether the RI-TATp53C′ peptide retained functionality, we compared the transducible parental L-isomer p53C′TAT and D-isomer RI-TATp53C′ peptides for the ability to induce a cell cycle arrest B. TreatmTo ascertain whether sustained arrest required the continuous presence of RI-TATp53C′ peptide, TA3/St cells were treated with peptide or vehicle for 2 d and then replated under mitogenic conditions in the absence of peptide. Peptide-treated tumor cells formed less than 1% as many colonies as vehicle-treated cells D. This oWe next investigated the ability of the RI-TATp53C′ peptide to transcriptionally activate p53-responsive genes. We transiently transfected p53 null human H1299 lung carcinoma cells with p53-dependent luciferase reporter plasmid (PG13-Luc) and either wild-type p53 expression plasmid or empty vector. The use of p53 null cells allows for negative controls that are not possible in cells expressing endogenous p53. As expected, we observed a p53-dependent induction of luciferase activity in cells transfected with p53 expression vector A. Howeve+/+p53 HCT116 colorectal carcinoma cells to HCT116 cells that were rendered p53-deficient at both loci by targeted genetic recombination (p53 HCT116 cells with RI-TATp53C′ peptide inhibited cell proliferation in a dose-dependent manner (+/+p53) and human Namalwa lymphoma cells that express a p53 hotspot DNA contact mutant (R248Q)  C. In con (R248Q) C. These  (R248Q) . Taken tWe  and othe3 by the end of treatment (p = 0.01) (We next tested the ability of IP administration of RI-TATp53C′ peptide to inhibit the growth of distant solid tumors in immune competent mice. Subcutaneous tumors in mice receiving either vehicle or mutant peptide grew rapidly, reaching an average volume of nearly 600 mmreatment B. In con = 0.01) B. These Because of their encapsulation and ectopic site of growth, subcutaneous tumors fail to replicate many of the features of terminal human cancer. We therefore tested the efficacy of RI-TATp53C′ peptide in a terminal peritoneal carcinomatosis mouse model that more closely resembles metastatic human disease. TA3/St carcinoma cells inoculated into the peritoneum of immune competent, syngeneic A/Jax (A/J) mice proliferated in a rapid logarithmic fashion, doubling in 24 h and increasing their numbers 100-fold 5 d postinoculation . This agp < 10−6). These observations demonstrate the ability of transducible peptides to significantly extend survival in a mouse model of terminal peritoneal carcinomatosis.We assayed the ability of the RI-TATp53C′ peptide to alter the tumor burden and increase the longevity of mice harboring TA3/St peritoneal carcinomatosis. Vehicle-treated mice rapidly succumbed to peritoneal tumor burden with a mean survival time of 11 d A. Mice tWe next investigated the biological consequences of peptide treatment to tumor cells in vivo. Peritoneal-TA3/St tumor-bearing mice were given daily injections of wild-type RI-TATp53C′ peptide or vehicle control. Mice were sacrificed 3 d after tumor cell inoculation for assessment of tumor burden. Vehicle-treated mice contained a significant tumor burden of recoverable dividing TA3/St tumor cells B. In conp < 0.0007)  B, with sThe subset of RI-TATp53C′ peptide-treated animals that succumbed to peritoneal carcinomatosis could have failed treatment either because of the emergence of peptide-resistant tumor cells or because of insufficient treatment duration. To distinguish between these two possibilities, we isolated TA3/St and Namalwa cells from animals that failed treatment. In both cases, the cells readily proliferated in culture under the same conditions as the parental cell population (data not shown). RI-TATp53C′ peptide treatment of reconstituted TA3/St cells induced a G1 arrest similar in extent to that of the parental cell line A. RI-TATAdvanced-stage peritoneal carcinomatosis and disseminated peritoneal lymphomas are often resistant to current chemotherapy treatment , and newThe vast majority of tumors express either wild-type p53 protein or a full-length p53 point mutant . This obThe work presented here provides several broad lines of evidence for the general feasibility of applying in vivo TAT-mediated transduction to cancer therapy. First, we find that inversion of the p53C′TAT peptide sequence and synthesis with D-amino acids results in a highly stable peptide (RI-TATp53C′) that retains both biological activity and the ability to transduce into cells. Given the rapid degradation of L-residue-containing peptides in vivo , use of Finally, most studies on anticancer transduction peptides have relied primarily on the use of solid, subcutaneous tumor growth as a measure of efficacy . AlthougCurrent clinical use of macromolecular biological therapies is limited to agents that have an extracellular mode of action. The preclinical data presented here demonstrate a proof-of-concept that intracellular delivery of biologically active macromolecular cargo by TAT-mediated transduction can modify specific pathways in vivo and that this approach potentially serves as a foundation for the generation of new classes of intracellular biological therapeutics.2. Short-term cell viability was assessed by counting Trypan blue-excluding cells on a hemocytometer. Long-term cell viability was assessed by colony formation assay. After serial dilution and 10 d of culture, colonies were washed in PBS and stained with 1% methylene blue. Cellular senescence was assessed by X-Gal staining as previously described (TA3/St (gift of W. G. Kaelin), H1299 (gift of R. K. Brachmann), and human foreskin fibroblast (M. Haas) cells were maintained in DMEM plus 10% fetal bovine serum (FBS) and penicillin/streptomycin (P/S). Namalwa cells (American Type Culture Collection, Manassas, Virginia, United States) were maintained in RPMI plus 10% FBS, P/S. HCT116 cells (gift of B. Vogelstein) were grown in McCoy's medium plus 10% FBS, P/S. All cells were maintained at 37°C in 5% COescribed , except Peptides were synthesized by standard Fmoc chemistry on an ABI 433A Peptide Synthesizer . Crude peptides were purified by reverse-phase HPLC over a C18 preparatory column . The identity of all peptides was confirmed by mass spectrometry.4 cells were plated per well. The next day, H1299 cells were transfected with 15 ng of TK-Renilla (Promega, Madison, Wisconsin, United States), 200 ng of PG13-Luc, and one of the following: 0.3 ng of empty vector, 0.3 ng of p53 expression vector, or 1 ng mutant of p53 expression vector (gift of R. K Brachmann). SW480 cells were transfected with 25 ng of TK-Renilla and 250 ng of PG13-Luc reporter plasmid. Cells were all transfected using Lipofectamine 2000 per the manufacturer's protocol . After 5 h, the transfection medium was removed and peptides were added to cells. Luciferase activity was measured 24 h later with the Dual Luciferase Reporter Assay System per the manufacturer's instructions (Promega).In a 96-well dish, 4 × 106 TA3/St cells in 200 μl of Hanks' balanced salt solution (HBSS). Tumor volume was estimated by V = (a2 × b)/2, where a is the short axis and b is the long axis of the tumor. IP TA3/St tumors were generated by injection of 2 × 106 TA3/St cells IP in 400 μl of HBSS. For the Namalwa lymphoma tumor model, 6- to 8-wk-old CB17 SCID female mice were obtained from Charles River Laboratory (Wilmington, Massachusetts, United States). Then, 5 × 105 Namalwa lymphoma cells were injected IP in 400 μl of HBSS. Peptide was dissolved in water, brought to 600 μl in PBS, and injected IP. All animal studies were approved by the University of California, San Diego, Institutional Animal Care and Use Committee.For TA3/St tumor models, 4- to 8-wk-old immune competent A/J female mice were obtained from Jackson Laboratory (Bar Harbor, Maine, United States). Solid TA3/St tumors were generated by subcutaneous injection of 1.5 × 10Mice harboring solid TA3/St tumors were injected with 650 μg of biotinylated RI-TATp53C′ peptide and sacrificed 1 h postinjection. Sections from frozen tumors were stained with Vectastain Elite ABC Kit and DAB substrate per the manufacturer's instructions .t-test was used to determine statistical significance (p < 0.05) in all experiments except animal survival experiments, in which the Wilcoxon Rank-Sum Test was performed.Student's