Macaca fascicularis) is one of the most widely used surrogate animal models for an increasing number of human diseases and vaccines, especially immune-system-related ones. Towards a better understanding of the gene expression background upon its immunogenetics, we constructed a cDNA library from Epstein-Barr virus (EBV)-transformed B lymphocytes of a cynomolgus monkey and sequenced 10,000 randomly picked clones.The cynomolgus monkey  were generated and assembled into 3,728 unigenes. Annotations of these uniquely expressed transcripts demonstrated that out of the 2,524 open reading frame (ORF) positive unigenes , 98.8% shared significant similarities  with the NCBI nucleotide (nt) database, while only 67.7%  did so with the NCBI non-redundant protein (nr) database. Further analysis revealed that 90.0% of the unigenes that shared no similarities to the nr database could be assigned to human chromosomes, in which 75 did not match significantly to any cynomolgus monkey and human ESTs. The mapping regions to known human genes on the human genome were described in detail. The protein family and domain analysis revealed that the first, second and fourth of the most abundantly expressed protein families were all assigned to immunoglobulin and major histocompatibility complex (MHC)-related proteins. The expression profiles of these genes were compared with that of homologous genes in human blood, lymph nodes and a RAMOS cell line, which demonstrated expression changes after transformation with EBV. The degree of sequence similarity of the MHC class I and II genes to the human reference sequences was evaluated. The results indicated that class I molecules showed weak amino acid identities (<90%), while class II showed slightly higher ones.These results indicated that the genes expressed in the cynomolgus monkey could be used to identify novel protein-coding genes and revise those incomplete or incorrect annotations in the human genome by comparative methods, since the old world monkeys and humans share high similarities at the molecular level, especially within coding regions. The identification of multiple genes involved in the immune response, their sequence variations to the human homologues, and their responses to EBV infection could provide useful information to improve our understanding of the cynomolgus monkey immune system. Macaca fascicularis) is one of the most widely used surrogate animal models for the studies of infectious diseases, organ transplantation, productive biology, and development of new vaccines.Non-human primates are ideal animal models for many human diseases because of their closely related genetic relationship and numerous biological and behavioral similarities with humans. As an important example, the cynomolgus monkey (in vivo HIV and other simian virus infections [Beyond a few sequences of the major histocompatibility complex (MHC) classical class I and II genes and cDNAs, at present little information is available about the genomic and gene expression background of the immune system of the cynomolgus monkey. Because the cynomolgus monkey serves as an ideal animal model for fections -5, HIV vfections , organ tfections ,8, tuberfections , and strfections , such knExpressed sequence tag (EST) projects provide a rapid and relatively efficient method for gene discovery, especially in organisms that have little information on genomics. Another advantage of using cDNA sequencing is that gene information is subjected to comparative genetic analysis among closely related species, for example, human and chimpanzee, which could greatly facilitate the evolutionary and genetic human studies, since the old world monkeys share high similarities with humans at the molecular level, especially within coding regions.DW522370-DW530304.Therefore, we adopted the EST strategy, sequenced and analyzed a collection of 8,312 ESTs from an Epstein-Barr virus (EBV) -transforLymphocyte cells were harvested and used to generate a non-normalized, directional cDNA library. Around 10,000 clones were randomly picked from the cDNA library and subjected to single-pass 5' sequencing using the T3 universal primer located at the up-stream of the vector backbone. After trimming low-quality and vector sequences and removing contaminant host sequences, a total number of 8,312 high-quality ESTs were obtained with a mean length of 509 bp. The length distributions of the ESTs are shown in Figure Gene-oriented clustering in EST analysis is often applied to the species that have had much sequence information on genes and/or genomes such as human and mouse. Although we have little such information of the cynomolgus monkey, the fact that great apes and humans share 98% similarities of their coding regions indicates that it is possible to use human mRNAs as references to direct gene-oriented clustering on cynomolgus monkey ESTs.Therefore, we downloaded all of the human mRNA reference sequences from the NCBI RefSeq database release 14)  and comp  and comBecause ESTs are single-pass sequences and are usually error prone, the introduction of reference sequences can produce more reliable results, especially for those cDNA libraries containing low-quality sequence data. We also assembled our ESTs using the original (non-oriented clustering) method and compared the results with that of gene-oriented clustering Figure . It showThe use of the reference sequences also enabled us to identify full-length sequences or full ORFs of our total 3,728 unigenes. BLAST searches against the human mRNA reference sequences indicated 3,128 unigenes matched to the database, among which 584  extended further upstream than the ATG (methionine) start codon of their homologs, including 305 contigs and 279 singlets. 4,864 full insert sequences of cynomolgus monkey cDNAs were also downloaded from NCBI and compared with our unigenes. The results yielded additional six full-length sequences, including one contig and five singlets. This relative low percentage of full-length inserts was mainly caused by the fact that the method we used was not optimized to generate a full-insert cDNA library. A summary of the alignment results is shown in Our following analysis was based on the results of gene-oriented clustering.Before the annotation process, the longest putative ORFs of all unigenes were determined by dynamically translating them in all six reading frames. Those unigenes containing too short ORFs (≤ 90 nucleotide bases or 30 amino acids) were considered non-informative ones and were subsequently excluded from the following analysis. The remaining 2,898 ORF positive unigenes  were compared with the NCBI nucleotide (nt) database and the NCBI non-redundant protein (nr) database using BLASTN and BLASTX, respectively. The searching results indicated that 183 unigenes were annotated to mitochondria encoded sequences, containing 2,768 ESTs; whereas 191 unigenes were annotated to genes coding for ribosomal proteins, containing 901 ESTs. Out of the remaining 2,524 unigenes , 2,493 (98.8%) had significant similarities  with sequences in the nt database, containing 3,691 (99.0%) ESTs; whereas 1,709 (67.7%) had significant similarities  with sequences in the nr database, containing 2,596 (69.6%) ESTs. Table Homo sapiens, Pongo pygmaeus, Pan troglodytes, and Macaca mulatta, which indicated that they could be the mostly expressed genes in these primates due to their numerous biological and genetic similarities.The annotation rate of the unigenes by BLASTX is surprisingly lower than that by BLASTN (67.7% versus 98.8%). With careful examinations, we found that 80.2% of the annotated unigenes  by BLASTN have best matches with mRNA and genomic sequences from closely related organisms, such as To identify new genes in cynomolgus monkeys, we compared the 784 unigenes that did not have significant matches to the nr database with public available 98,325 ESTs of cynomolgus monkeys downloaded from the NCBI dbEST database  using BLFor the 75 unigenes that matched to the human genome, we further analyzed their aligned regions by comparing the BLAT results with the refGene database from UCSC . They feThe comparative results could be very helpful for us to discover more human genes that are currently not present in the human genome annotation reservoir and to revise those incompletely or incorrectly annotated ones by using genes from cynomolgus monkeys as probes and primers. The old world monkeys are highly similar to humans at the molecular level, especially within coding regions. For example, we share over 98% of our genes with apes. By comparing the novel transcripts in cynomolgus monkeys with the human genome and mapping these transcripts to human and chimpanzee chromosomes, we could isolate new genes that may not be present in current gene annotation reservoirs of both species.de novo methods can correctly predict only 70% of individual exons and all the coding exons of 20% genes in the human genome, with a large number of false-positives [ab initio gene-finding programs could provide high enough specificities and outperform those purely gene prediction methods [Another advantage that we can take from genes expressed in cynomolgus monkeys is to improve the overall performance of gene-finding programs by comparative methods. Currently, ositives . The sit methods -21.Because the cDNA library was derived from transformed B cells without normalization, it represents a gene expression profile at idealistic environments. By analyzing all unigenes for their functional characteristics using the Gene Ontology (GO) , we can The largest subcategory found in "biological process" was physiological process, consisting 87.4% of the unigenes in this category. The second largest subcategory was cellular process, consisting 87.0% of the unigenes. These abundantly expressed genes indicated that cells were undergoing rapid growth and metabolism, which is consistent with the materials that the cDNA library was derived from. Other highly abundant subcategories including binding and catalytic activity in "molecular function" also confirmed this conclusion.The GO function categories for our library were further compared with a human B cell  cDNA library (dbEST Library ID.13050) downloaded from the NCBI UNIGENE database Figure . The BurCareful examinations of the genes involved in biological process of these two cDNA libraries Figure  revealedst, 2nd and 4th most abundantly expressed protein families were all immunoglobulin-related, indicating that our cDNA library is suitable for the discovery of genes involving in antigen representations and recognitions, protein-protein and protein-ligand interactions, which could improve our basic understanding of the immune system of the cynomolgus monkey.The latest InterPro  databaseGenetic studies have revealed that MHC associates with more human diseases than any other regions of the human genome, including most of the immune-related disorders, for example, insulin dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), ankylosing spondylitis (AS), common variable immunodeficiency (CVID) and IgA deficiency (IgAD) . Strong The using of the cynomolgus monkey as a surrogate animal model has greatly facilitated the developments of new medicines against human diseases, especially those immune-related ones such as HIV and heart transplantation. But the lacking of knowledge on the MHC region of the cynomolgus monkey has become a bottleneck to basic genetic researches and bio-medical industries.By constructing the cDNA library using B lymphocytes that play important roles in antigen-representation in the immune system, we expected as many as possible genes, especially MHC-related ones. In our cDNA library, totally 39 unigenes were annotated to MHC antigens using BLASTX, including four MHC class I genes: MHC_A, B, E and F, eight MHC class II molecules, and five ones homologous to HLA-related genes: HLA_B associated transcript 2 (BAT2), complement component 4B (C4B), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like (NFKBIL1), valyl-tRNA synthetase and casein kinase 2, consisting 43, 129 and 14 ESTs, respectively.To evaluate the degree of sequence similarity of the MHC class I and II genes between cynomolgus monkeys and humans at both nucleotide and protein levels, we analyzed the overlapped sequences with the coding sequences of their corresponding human references. The overall protein similarities were slightly higher than that of nucleotides Table , indicatThe relative expression abundances of the MHC-related genes as transcripts per million were calculated and compared with those in the RAMOS cell line and two most related human tissues to B cells: blood and lymph node Figure . Since MLymphocytes can be divided into subsets either by their functions or by surface markers that have been designated as Clusters of Differentiation (CDs), which play various but central roles in B cell activation, development and immunological response regulation. Currently, as many as 356 CDs  have beeCD19 is a B-cell specific molecule that regulate B cell development, activation, differentiation and antigen receptor-mediated MHC class II antigen processing . The memCD74 is the most abundant one among the surface markers, which contained 78 ESTs , comparing with 11,533 transcripts per million ESTs in the human lymph node. CD74 plays an important role in the antigen representation by acting as a specific chaperone in MHC class II assembly and transport  and was After infection by EBV, B cells were immortalized and some tumor-like features would appear. Several CD members indicated such state of abnormity. For example, CD147 , the second most abundant CD in our cDNA library, is a major mediator of malignant cell behavior in the human, and is regulated by several membrane-associated cofactors, including annexin II (containing one EST) and caveolin-1 . These sMacaca mulatta) [Besides MHC genes and many CDs, several other immunoglobulin genes were identified in our library, including immunoglobulin alpha heavy chain constant region , immunoglobulin kappa-chain and immunoglobulin lambda-chain. Recent studies showed that alleles of a single immunoglobulin C alpha chain were presented in rhesus macaques (mulatta) , indicatAlthough with the absence of some key molecules in the activation of immune responses to facilitate the virus transformation, the transformed B lymphocytes from the cynomolgus monkey expressed more immunoglobulin and ligand and receptor genes than in human blood and lymph nodes, in numbers as well as in abundances; A B-cell specific proliferation pathway including MIF, CD74 and CD44 was greatly enhanced after transformation. These results indicated that this cDNA library represents an ideal model to improve our understanding of mechanisms of antigen representations and immune responses of cynomolgus monkeys. The differentially expressed genes between our cDNA library and normal B cells of cynomolgus monkeys, if available, should be in great interests, because of their possible key functions corresponding to the immune response and malignant cell growth. Yet one of the greatest challenges is that we do not know which one of the differentially expressed genes is the reason of the transformation and which one is just the result. Much more researches are needed to be done until we get the answer.We have constructed a cDNA library from lymphocytes of the cynomolgus monkey and sequenced about 10,000 randomly picked clones. Approximately all (98.8%) ORF positive unique transcripts  shared significant similarities with the NCBI nt database, but only 67.7% of these transcripts shared significant similarities with the NCBI nr database using BLAST-based tools. Further analysis revealed 420 new transcripts in cynomolgus monkeys; in which 139 did not share significant similarities with any human ESTs. Such sequence information was used in comparative analysis to identify possible novel genes in the human genome.The cynomolgus monkey is one of the most widely used surrogate animal models for an increasing number of human diseases and vaccines, such as HIV and heart transplantation. But the lacking of background information at the genomic level especially in the immune system has greatly hinged our progress. Our cDNA library, which was derived from the EBV-transformed B lymphocytes of the cynomolgus monkey, could identify as many as possible immune-related genes and reveal the gene expression profile. Since many human diseases are associated with the MHC region and deficiencies of immune system, such knowledge is of great interest to researchers and industries that use cynomolgus monkeys as experimental models.et al [An amount of 4–5 mL whole blood was sampled from a cynomolgus monkey, followed by the isolation of lymphocyte cells. The B-lymphocyte cell line was established according to Scammell et al .® II XR vector (Stratagene) exploiting the EcoRI and XhoI restriction sites according to the manufacturer's instruction.Total RNA was extracted from culturing cells using the Trizol agent (Invitrogen) according to the manufacturer's instruction. mRNA was subsequently purified using the mRNA PolyATtract@mRNA isolation system (Promega). The cDNA library was constructed in the directional pBluescriptE. coli) and valid average (masked) length. The randomicity of the cDNA library was determined by calculating the ratio of unique sequences (contigs + singletons)/reads.The quality of the cDNA library was first assessed by colony PCR of 96 randomly picked clones to determine the average insert size and percentage of clones without inserts. Then 384 randomly picked clones were sequenced to determine the ratio of contaminations , grown overnight on solid LB medium containing IPTG and X-Gal. 10,000 colonies were picked, grown overnight in LB medium containing Amp in 96-well plates. An amount of 5 μL from each well were transferred into 384-well microtitter plates containing 5 μL 20% glycerol (V/V) to preserve a permanent clone stock. Plasmids were isolated and sequenced on MegaBase® 1000 sequencers using the T3 universal primer that anneals to the plasmid backbone upstream of the 5' end of the cDNA inserts.Plasmid constructs were transformed into Phred ,43 was uHuman mRNA sequences from the NCBI RefSeq database were downloaded and used as reference sequences to direct clustering. Firstly, the reference sequences were compared with our ESTs using BLASTN with an E-value≤1e-6. ESTs that aligned to the same mRNA were assembled along with the mRNA using the phrap program with more strict parameters (-minmatch 40 -minscore 60) to distinguish possible individual members of possible multi-gene families.The lymphocyte unigenes were compared with the NCBI nt database using the BLASTN program and with the protein sequences in the NCBI nr database using the BLASTX program. They were also compared with public available full-length inserts as well as cynomolgus monkey and human ESTs. The E-value cutoffs were 1e-10 for BLASTN and 1e-5 for BLASTX, respectively. Functional classes were assigned according to GenBank to GO mapping provided by the GO website. Then the distributions of the gene families and functional domains were assigned according to the sequence similarities of our unigenes to the InterPro database.SNH and XNW participated in designing the research; XXW, XWH, ZQW, WL and YL performed the research; WHC controlled and analyzed the data; WHC and XXW wrote the paper.Statistics of BLAST searches against the human reference sequences (RefSeq). The coverage was calculated by dividing the length of HSPs (high scoring pairs) by the CDS length of aligned references. Out of the 3,728 unigenes, 3,128 matched to the database.Click here for fileThe GO assignment results of the cynomolgus cDNA library.Click here for file


The generation of hydrogen peroxide (H2O2) and the derivative free hydroxyl radical (. OH) in cultures of mouse cells grown in the presence of visible light and ambient oxygen was shown previously to be implicated in chromatid damage. Furthermore, chromosome alterations appear to be associated with the spontaneous neoplastic transformation of mouse cells in culture. An attempt was made in this study to reduce the incidence of chromosomal aberrations and delay or prevent the onset of spontaneous neoplastic transformation of freshly isolated mouse cells, both fibroblasts and epidermal keratinocytes, by adding catalase to the culture medium, shielding the cultures from wavelengths less than 500 nm and providing a gas phase of 0-1% O2. These conditions significantly decreased the incidence of chromosomal aberrations in both cell types, and in fibroblasts prevented tumourigenicity in non-irradiated syngeneic mice, and increased latent periods for tumour development in X-irradiated mice. The epidermal keratinocytes were particularly resistant to spontaneous neoplastic transformation under all conditions tested. These observations on the protective effect of extracellular catalase suggest that H2O2, a normal metabolite, and/or the derivative .OH can directly or indirectly produce genetic damage and neoplastic transformation in mouse fibroblasts.


Air pollution remains a leading cause of many respiratory diseases including chronic cough. Although episodes of incidental, dramatic air pollution are relatively rare, current levels of exposure of pollutants in industrialized and developing countries such as total articles, diesel exhaust particles and common cigarette smoke may be responsible for the development of chronic cough both in children and adults. The present study analyses the effects of common environmental factors as potential causes of chronic cough. Different PubMed-based researches were performed that related the term cough to various environmental factors. There is some evidence that chronic inhalation of diesel can lead to the development of cough. For long-term exposure to nitrogen dioxide (NO2), children were found to exhibit increased incidences of chronic cough and decreased lung function parameters. Although a number of studies did not show that outdoor pollution directly causes the development of asthma, they have demonstrated that high levels pollutants and their interaction with sunlight produce ozone (O3) and that repeated exposure to it can lead to chronic cough. In summary, next to the well-known air pollutants which also include particulate matter and sulphur dioxide, a number of other indoor and outdoor pollutants have been demonstrated to cause chronic cough and therefore, environmental factors have to be taken into account as potential initiators of both adult and pediatric chronic cough. Coughing and mucus secretion are coordinated neuronal reflexes that protect the respiratory tract from noxious exogenous substances under physiological conditions. However, within chronic exposure to noxious substances such as tobacco smoke, urban dust, or occupational factors -3, the oThese suggestions do not only base on epidemiological and clinical observations, but also on the neurophysiological and -anatomical understanding of the cough reflex. In this respect it is generally accepted, that airway pollutant-caused airway irritation leading to chronic cough displays a complex phenomenon involving a variety of reflex mechanisms -19.Neurophysiologically, a subgroup of rapidly adapting receptors (RARs) among the three major types of vagal sensory receptors is suggested to act as "cough receptors". Next to these RARs, a further effect by bronchopulmonary C-fibers on the cough reflex has been suggested, and there are data indicating that i.e. ozone, one of the main environmental air pollutants, exerts an influence on vagal-sensory innervation -24. AlsoIn the light of the clinical, epidemiological and experimental data which point to a major role of environmental pollutants as co-factors for the development and progression of chronic cough, the present study analysed the data available on the association between environmental pollutants and chronic cough on the basis of a large amount of existing recent literature reviews and original articles ,13,34-55A PubMed research was performed using terms such as "cough", "environmental", and various environmental factors and publication types (date: 2006-03-03). Articles were screened for their contents and relevant data was analysed.For the terms „cough”and journal article as publication type 23248 entries were registrated in the PubMed while 2463 review articles containing the term cough were found  for diesel emission exposed individuals varied between 1.2 and 2.3 and there was a consistent finding for an increased risk of chronic cough among the studies -59. In gNitrogen dioxide (NO2) is an oxidizing free radical which can initiate a number of destructive pathways in the human body ,60. NO2 In 1985, primary schoolchildren and their mothers were surveyed in Hong Kong to study the possible relationship of nitrogen dioxide (NO2)-related environmental air pollution to respiratory illnesses . Using pThe effect of indoor nitrogen dioxide exposure on the incidence of respiratory symptoms and pulmonary function level was also examined in an American cohort of 1,567 Caucasian children aged between seven and eleven years and examined from 1983 through 1988 . Nitroge3 in pollutant levels was associated with an increase in the prevalence of chronic cough [Also, the SAPALDIA study examined the association between NO2 and chronic cough and it was reported that an increase of 10 μg/mic cough . Next toic cough  also repic cough .Together, these data indicate that long-term NO2 exposure leads to increased incidences of chronic cough and decreased lung function. Also, weak associations between short-term NO2 exposure and respiratory symptoms and a decrement in lung function parameters were found in children, but not consistently in exposed women.With regard to one of the most dramatical urban environmental exposures to air pollutants, the London smogs -70, sulpA study on indoor air pollution and respiratory health in urban and rural China revealed interesting relations between chronic cough and SO2 : During A further study in Canada also addressed the association of inhalable sulfates, SO2 and childhood chronic cough : PreadolOzone is a very powerful oxidant and also a very toxic air pollutant ,74. As aA variety of studies focused on the relation between ozone-exposure and chronic cough Fig.  and reveIn the cross-sectional study in Canada with preadolescent school children, aged 7–11 years, in the low-exposure region central Saskatchewan, and in the moderately polluted area in southwestern Ontario, no significant regional differences were observed in rates of chronic cough. Here, the annual mean of the 1-hr daily maxima of ozone was higher in Ontario (46.3 ppb) than in Saskatchewan (34.1 ppb), with 90th percentile concentrations of 80 ppb in Ontario and 47 ppb in Saskatchewan and summertime 1-hr daily maxima means were 69.0 ppb in Ontario and 36.1 ppb in Saskatchewan .In the Swiss SAPALDIA and SCARPOL studies, no associations between ozone and chronic cough were found ,65.In contrast to these epidemiological data, there is a large body of experimental evidence that ozone influences pulmonary vagal-sensory nerve fibers which are suggested to be major mediators of the cough reflex ,80. HoweIn this respect, exposure-response relations are non-linear for the respective associations between O3 and FEV1, inflammatory changes, and changes in hospital admissions, whereas it has been reported that the relation between percent change in symptom exacerbation among adults and asthmatics is linear. Also, the single-pollutant associations between ozone exposure and hospital admissions for respiratory diseases and daily mortality were shown to be statistically significant, even in multi-pollutant models.There is also a variety of other environmental factors which have been reported to contribute to the prevalence of chronic cough. In this respect, recent reports have demonstrated a relation between allergen such as Humicola fuscoatra in indoor air and chronic cough (combined with sputum eosinophilia)  or arsenAlso, depleted uranium, which is a radioactive heavy metal that is commonly used in missiles, has been shown to be associated with chronic cough as a health risk .Next to microbially-related chronic cough , a furthEnvironmental air pollution is not only one of the causes that might lead to severe diseases such as cancer or cardiorespiratory disease, but also associated with the prevalence of chronic cough both in adults and children.Although episodes of incidental, dramatic air pollution are relatively rare, the currently found levels of exposure to pollutants especially in developing countries may increase the prevalence of chronic cough. Next to well-known air pollutants such as nitrogen dioxide, a number of other indoor and outdoor pollutants have recently been demonstrated to cause chronic cough and therefore, environmental factors have to be taken into account as potential initiators of both adult and pediatric chronic cough.The author(s) declare that they have no competing interests.BGK and AF have planed the study. BGK has peformed the data analysis and interpretation and drafted the article. TK, AVM and UW have all been involved in interpreting and discussing the data and revising the article critically for important intellectual content.


In order to examine in detail the sensitivity to chemotherapy of tumour cells at various organ sites and at various stages of metastasis, we have used a series of cell lines, all selected from sister subpopulations derived from a single mouse mammary tumour, which can be distinguished and quantitated from normal cells and from each other through growth in selective medium. For the studies described here, we used two lines, 4T07 and 66FAR, which will form colonies in vitro in medium containing 60 microM 6-thioguanine or 330 microM 2,6-diaminopurine, respectively. Both cell lines have similar sensitivity to the test chemotherapeutic agent, melphalan, in vitro. These two tumour cell lines were treated with melphalan in vivo, when growing either in lungs as experimental metastases at various times after cell injection or as palpable tumours growing subcutaneously. Responses to various doses of melphalan were measured by removing lungs or subcutaneous tumours and performing colony-forming assays in selective medium. The data indicate marked shifts in sensitivity as a function of metastatic stage. Analyses of dose-response curves show that both cell lines were similarly sensitive to melphalan at early times (45 min) after cell injection i.v. but became less sensitive at an intermediate time after injection (3 days). Differences between the two lines became apparent at later times after i.v. injection (by day 8 or 9) and in subcutaneous tumours, where a marked reduction in the shoulder of the dose response curve was seen in line 4T07, resulting in sensitivity equal to or greater than the of early times, whereas the dose response parameters of 66FAR remained at those of the intermediate time point. These results show that, in heterogeneous tumours, individual subpopulations of tumour cells may respond differently to chemotherapeutic agents at various disease stages. In vitro measures of tumour sensitivity do not predict these changes in in vivo sensitivity. Model systems similar to the one described here may yield information which will eventually be useful in maximising the efficacy of clinically relevant adjuvant chemotherapy regimens.


The expression of pS2 was examined histochemically in paraffin sections taken from biopsy material from patients diagnosed with ductal carcinoma in situ (DCIS). Often intense immunoreactivity, to an anti-pS2 monoclonal antibody, was observed in comedo, solid, cribriform and micropapillary types of DCIS, with significant positivity found in 63-67% of cases. In 15 samples analysed, we found a good correlation between pS2 expression and presence of progesterone receptor positive cells, but not with estrogen receptor. There was only a limited degree of correspondence between the cells staining with these anti-sera. Some pS2 positive cells were also seen in normal acini in areas adjacent to cancer but much less frequently in sections of normal breast from reduction mammoplasty. Most normal areas were negative, as were cysts. In benign proliferative conditions (seen in sections with and without DCIS) such as adenosis, sclerosing adenosis, mild and florid ductal epithelial hyperplasia, significant pS2 positivity was seen in about 50% of cases. These results suggest that there is a progressive increase in pS2 from normal to benign to cancer cells and that this gene is expressed in both the invasive and pre-invasive forms of breast cancer.


A systematic study of the transcript variants of all protein kinase- and phosphatase-like loci in mouse shows that at least 75% of them generate alternative transcripts, many of which encode different domain structures. .Alternative transcripts of protein kinases and protein phosphatases are known to encode peptides with altered substrate affinities, subcellular localizations, and activities. We undertook a systematic study to catalog the variant transcripts of every protein kinase-like and phosphatase-like locus of mouse By reviewing all available transcript evidence, we found that at least 75% of kinase and phosphatase loci in mouse generate alternative splice forms, and that 44% of these loci have well supported alternative 5' exons. In a further analysis of full-length cDNAs, we identified 69% of loci as generating more than one peptide isoform. The 1,469 peptide isoforms generated from these loci correspond to 1,080 unique Interpro domain combinations, many of which lack catalytic or interaction domains. We also report on the existence of likely dominant negative forms for many of the receptor kinases and phosphatases, including some 26 secreted decoys  and 13 transmembrane forms . Finally, by mining public gene expression data (MPSS and microarrays), we confirmed tissue-specific expression of ten of the novel isoforms.These findings suggest that alternative transcripts of protein kinases and phosphatases are produced that encode different domain structures, and that these variants are likely to play important roles in phosphorylation-dependent signaling pathways. The results of the 5'-RACE analysis and the primer sequences used are provided in Additional data file 5.To test whether the threshold of counts we applied was biologically relevant and whether cDNAs starting from within internal exons of longer transcripts are 5' truncations or genuine transcription start sites, we tested a panel of 19 alternative 5' exons with 5'-RACE. As a technical point, an enzymatic oligo-cap method independent of the FANTOM3 cap-trapper technique was used to ensure that only full-length capped 5' ends of mRNAs were surveyed ,30. PredThe analyses described above used all available cDNA evidence, with many variants only detected as partial EST sequences. Although ESTs provide a deeper sampling of alternative transcripts, interpretation of variants found in these sequences is confounded by their bias to the termini of transcripts (due to EST sequence generation providing short reads coming from 5' and 3' termini of cDNAs) and problems associated with sequence quality arising from single sequencing reads for each EST. We therefore chose a more conservative approach and used only full-length cDNAs to examine alternative peptides encoded from these loci.A total of 5,877 phosphoregulator full-length transcripts from FANTOM, GenBank, and RefSeq were filtered based on the following: redundant entries that shared the same splice junctions, TSS, and TTS were removed; transcripts with stop codons more than 50 bases upstream of their final splice junction were excluded as NMD candidates  . Variant peptides were then classified into the following four classes: 582 peptides with the full complement; 147 variants with disrupted or missing accessory domains; 161 variants with disrupted or missing catalytic domains; and 190 with disruptions to both accessory and catalytic domains . These classifications were then added as annotations in the web interface. A list of all variants detected is provided in Additional data file 11. In Tables A class of phosphoregulators with multiple reported examples of transcriptionally derived dominant negative products is the receptor kinases. For these loci, multiple soluble secreted and membrane-tethered decoy receptors lacking catalytic domains have been described. We therefore undertook a computational review of transcripts of the 56 tyrosine receptor kinase, 12 serine/threonine receptor kinase, and 21 tyrosine receptor phosphatase loci of mouse to determine their potential to generate dominant negative gene products.Conceptually, receptors are divided into two parts: the extracellular ligand-binding portion of the peptide and the intracellular catalytic portion. Signal peptide and transmembrane domains are both required for correct targeting and anchoring of type I membrane peptides within the plasma membrane. Each transcript variant was reviewed for changes in the predicted peptide that would affect localization signals or catalytic domains.We identified two classes of ORFs encoding catalytically inactive variant peptides predicted to compete for ligand in the extracellular space Table : 13 poten = 13), whereas others lacked both the signal peptide and the transmembrane domain (n = 12). We refer to these two variant types as 'TMcatalytic' and 'catalytic', respectively. TMcatalytic forms resemble the type 2 transmembrane phosphoregulators such as the nonreceptor phosphatase Ptpn5, which localizes to the endoplasmic reticulum [The review of these loci also identified a further two classes of potential variants. Alternative TSS within loci frequently generated transcripts encoding peptides that lacked amino-terminal features. Many of these variants lacked the signal peptide . All but two of the secreted and tethered forms are generated by alternative 3' ends hence we searched for microarray probes and MPSS  signatures diagnostic of these alternative 3' ends. The Mouse Transcriptome Project (trans-NIH with Lynx MPSS™ technology) provides MPSS gene expression data from a panel of 85 tissue samples ,34. SimiMPSS also provided evidence for tissue-specific expression of nine novel isoforms: seven secreted forms ; one tethered form of Axl in kidney; and one catalytic form of Ptprg in brain, kidney, white fat, and cartilage. Similarly, the GNF gene atlas provided evidence for tissue-specific expression of two novel secreted isoforms: Ptprk in blastocysts and Ptprg in brain. For the catalytic and TMcatalytic forms of Ptpre and Ptpro, CAGE tags confirmed their reported restriction to the macrophage lineage ,38.As part of this review, we identified four novel transcripts for the colony stimulating factor 1 receptor Csfr1. Three of these transcripts were predicted to encode potential tethered isoforms, whereas a fourth encoded a potential secreted version of the receptor Figure .In order to determine the likelihood of efficient expression and subcellular targeting of these novel variants, we undertook transient expression assays of the Csf1r variants in mammalian cells and confirmed that the truncated tethered forms are targeted, as predicted, to the plasma membrane whereas the form lacking the predicted transmembrane domain exhibits a secretory pathway-like localization Figure .AK155565) was detected at levels similar to those of the full-length receptor . A role for these variants in cell migration is supported by observations for Epha7 variants and the catalytically inactive Ephb6 [We also reported probable dominant negative forms for eight of the 14 Eph receptors in mouse  and a review of sequences from other species revealed probable dominant negative forms for three of the remaining six  and the insulin receptor related genes . Alternative splicing of exon 11 of the insulin receptor in human has previously been reported , but no Proteolytic processing for many of these receptors split the protein into a soluble extracellular fragment that is capable of binding ligand and an intracellular catalytic fragment   are knowAnother class of variant transcripts is predicted to undergo NMD. Using the '50 base rule', transcripts with premature termination codons more than 50 bases upstream of a final exon junction were filtered out as NMD candidates targeted for destruction . HoweverDespite this, a number of the transcripts that break the 50 base rule still appear to represent full length messages with short predicted introns in their 3' untranslated regions. We identify four loci Rps6ka4, Map3k1, Epha4, and Pxk that have predicted final introns in their 3' untranslated regions of 126, 1555, 3239, and 114 bases, respectively. All NMD predictions are provided in Additional data file 8 and online .In cases in which peptide variations disrupt or remove an accessory domain, constitutively active -64 or doThese variants not only add to the peptide diversity of the phosphorylation system, but they are also intrinsically related to the function of all peptides generated from the same locus. They are likely to compete for the same ligands and substrates, but by changes in the peptide their activity, stability, localization, and regulation may be altered. This opens up the possibility that transcriptional control of the mix of isoforms present within a system is used as an additional mechanism to regulate the overall status of the system.Regulated use of alternative promoters, terminators, and splice junctions allows a cell to produce either alternative peptides with slightly different activities or the same peptide in a different context. In some cases these choices are 'hard wired' during differentiation, such that one isoform is produced in a particular cell type  whereasSystematic analysis of every protein kinase and phosphatase of mouse has revealed that for most of these loci alternative transcripts are generated. The use of alternative transcription initiation, termination, and splice junction sites offers three mechanisms for controlling the functional output of the locus. We provide evidence for alternative 5' and 3' end usage and document a large set of variant peptides and domain structures. Finally, we suggest that, for complete understanding of signal transduction and protein phosphorylation in general, these forms must be considered components of the network and that regulation of these forms in development and on challenge indicates a fundamental coupling of transcriptional control with protein phosphorylation.For each locus a three frame view combined genomic and transcript centric views from FANTOM3 ,74 with The 5' and 3' ends of full-length cDNA, ESTs, and tag sequences from CAGE , GenomicMus musculus 5, Mouse genome assembly, build 33) assembly of the mouse genome sequence (May 2004) [Conceptually, two levels of clustering were carried out to provide end support. Tag clustering grouped transcripts that shared TSS or TTS based on the overlap of their termini (20 bases) relative to the UCSC mm5 (ay 2004) . Exon clThe genomic mappings of every multi-exon cDNA and EST were extracted from the FANTOM3 analysis ,76. ExonThe nucleotide sequences of the probes used in the GNF gene atlas arrays and the MPSS signature sequences were aligned to transcript sequences using BLAST  ,35. DiagNMD predictions were made by calculating the distance between the last splice site and the stop codon of full length predicted. Splice sites were determined by alignments to mm5. A total of 191 sequences for which the final splice site was more than 50 bases from the stop codon were flagged as putative NMD targets . A numbeFor each locus, InterProscan predictions were used to assess changes in domain content of each variant . Using tcDNA clones of variant Csf1 receptor  were subcloned into a mammalian expression vector. HeLa cells were transiently transfected for 16 hours, formalin fixed, and processed for immunofluorescence. Recombinant Csf1r was detected using the rat monoclonal AFS98 antibody .RNA was harvested from cells using the RNeasy kit . First strand synthesis was carried out on 1 μg total RNA using Superscript III . Real-time PCR was performed with the SYBR qPCR SuperMix-UDG kit (Invitrogen). Twenty microliter reactions were performed in an ABI 7700 , with 35 cycles of 1 minute elongation at 60°C; all reactions were performed in duplicate. Relative fold change of full length and variant were calculated using the delta Ct (cycle threshold) method.5'-RACE experiments were performed using an enzymatic oligo-capping method  that ensThe following additional data files are available : an ExcAn Excel file listing all protein kinase-like and protein phosphatase-like loci considered in this study .Click here for fileAn Excel file listing alternative splice junctions identified in the set and the cDNA accession numbers that support them.Click here for fileA zip file containing four Excel files .Click here for fileA zip file containing a PowerPoint presentation with genomic views of the 5'-RACE results and an Excel file summarizing the results and the primer sequences used.Click here for fileAn Excel file of zinc finger loci with levels of support for alternative transcripts.Click here for fileAn Excel file that contains supporting evidence for the variant receptors discussed in the results, providing links to MPSS, GNF, and CAGE for transcriptional evidence, links into PubMed for known examples, and other supporting evidence.Click here for fileA pdf file containing a listing of clones predicted as NMD candidates.Click here for fileAn Excel file containing the domain combinations, complements, and raw Interpro results for all full-length transcripts in the phosphoregulator set.Click here for fileA pdf file showing a graph of the number of loci with alternative splice junctions, and 5' terminal or 3' terminal exons .Click here for fileAn Excel file summarizing the predicted domain combination and variant type for the 1473 full-length ORFs identified in the domain structure analysis.Click here for fileA zip file containing an Excel file summarizing the quantitative real-time PCR results for the Csf1r receptor variants and a pdf file containing additional localization images for the secreted isoform.Click here for file


Drosophila. Here we report that in the epidermis of the Drosophila embryo two signaling molecules, Hedgehog (Hh) and Wingless (Wg or Wnt1), provide directional cues that induce the proper orientation of Actin-rich structures in the larval cuticle. We further find that proper polarity in the late embryo also involves the asymmetric distribution and phosphorylation of Armadillo (Arm or β-catenin) at the membrane and that interference with this Arm phosphorylation leads to polarity defects. Our results suggest new roles for Hh and Wg as instructive polarizing cues that help establish directionality within a cell sheet, and a new polarity-signaling role for the membrane fraction of the oncoprotein Arm.To generate specialized structures, cells must obtain positional and directional information. In multi-cellular organisms, cells use the non-canonical Wnt or planar cell polarity (PCP) signaling pathway to establish directionality within a cell. In vertebrates, several Wnt molecules have been proposed as permissible polarity signals, but none has been shown to provide a directional cue. While PCP signaling components are conserved from human to fly, no PCP ligands have been reported in  Drosophila and hair patterns and inner ear epithelia in vertebratesCaenorhabditis elegans Wnts have been implicated in cell polarity, both in asymmetric divisions in the early embryo and in the direction of neuronal polarizationDrosophila, Wnts have largely been excluded from polarity signalingCells in multicellular organisms must establish directionality within the plane of a sheet of cells (planar cell polarity or PCP) in order to form complex structures such as organized wing hairs and photoreceptors in Drosophila embryo establishes a segmented body planAnterior-posterior patterning in the Drosophila oogenesisin vivo requirement for threonine phosphorylation of Arm that regulates Arm at the adherens junction. This threonine phosphorylation is thought to stabilize the interaction between α- and β-catenin, thereby leading to stabilization of cellular junctionsArm protein is both the major nuclear effector of Wg signaling and a major component of adherens junctions. Adherens junctions provide much of the cell-cell adhesion in epithelia through the transmembrane Cadherin molecules, which establish a physical link between cells. The intracellular domain of Cadherins recruits β-catenin, which in turn recruits α-catenin, thereby linking the transmembrane junctions to the Actin cytoskeletonDrosophila larvae prompted our investigation of how the Hh and Wg signaling pathways and their components are involved in this polarity. Here we demonstrate that Hh and Wg ligands provide opposing, instructive signals for the orientation of denticles. By genetically manipulating the direction of ligand expression in relatively naïve epithelia, we observe a rotation of polarity consistent with the direction of ligand expression. Further, we find a phosphorylation-dependent role for Arm in the establishment of planar polarity through its function in the polarized subcellular localization of denticle precursors. These results identify new roles for Wg, Hh, and Arm in organizing PCP in embryonic epithelia.The polarized organization of the epidermis of Drosophila embryos where loss of junctions leads to cuticle disintegration and loss of nuclear signaling leads to patterning defects (compare AA) that are required in vitro to stabilize its binding to α-cateninDrosophila cuticles to analyze the effect of ArmAA on both the adherens junctions and on Wg signaling in the nucleus. In strong loss-of-function O43A01arm maternal and zygotic (M/Z) mutants (hereafter referred to as O43A01arm (M/Z)) no intact cuticle is made because loss of Arm leads to a drastic loss of cell-cell adhesion in the epidermis. O43A01arm (M/Z) embryos eventually disintegrate and only small pieces of tissue remain (AA expression restored some cuticular integrity that is completely lost in O43A01arm (M/Z) embryos, suggesting restoration of some cell-cell adhesion in the epidermis  mutants, which restores adherens junctions by bypassing the need for Arm to bridge the α-catenin and E-cadherin interaction. However, expression of this fusion protein does not restore Arm's nuclear signaling function as indicated by a lack of anterior-posterior patterning, or more specifically, a loss of naked cuticle and a lawn of denticles phenotypeAA expression levels restored some nuclear Wg signaling activity, as indicated by partial restoration of anterior-posterior patterning, which is shown here by the presence of both naked cuticle and cuticle with denticles  mutants, the truncated ArmXM19 protein does not activate the Wg signal in the nucleus, but does retain function in the adherens junctionAA in XM19arm (M/Z) mutants lead to an essentially wild-type cuticle in terms of both patterning and denticle organization,  double mutant) leads to a completely naked cuticle, a hallmark of constitutive Wg signaling activation . In contrast, in O43A01arm (M/Z) embryos expressing ArmAA, only about half of the denticles point correctly (48.0±5.6).To further demonstrate that this disorganized denticle phenotype was a defect in planar organization; we compared the orientation of denticles in various genotypes. As described above, wild-type denticles are highly organized and almost always point toward the anterior or posterior of the embryo  and 2A. AA embryos, the Actin foci are localized randomly  mutant embryos that expressed Hh along the ventral midline. As expected for standard Hh signal transduction, in the absence Smo, Hh cannot affect the rotation of denticles because the Smo/Ptc receptor complex is compromised , repression of the Hh pathway activator Smoothened (Smo) is relieved, thereby activating the Hh pathwaypromised [13].disheveled (dsh M/Z) null mutant embryos and expressed Wg along the ventral midline. In Wg signaling, the Dsh protein is required for the transmission of Wg signal within cells, and acts in both the PCP and canonical Wnt pathways. Embryos lacking Dsh resemble wg mutants in both the lack of naked cuticle and the lack of denticle organization  is required for Wg signaling in the nucleus. In the absence of TCF (pan mutants), expression of Wg along the ventral midline had no visible effect on the polarity of the denticles (results summarized in cubitus interruptus (ci or Gli). When we expressed Hh along the ventral midline in ci mutants, we observed that Hh caused no visible effects. These results taken together suggest that both the Hh and Wg ligands require a transcriptional response in order to affect denticle polarities.Next, we examined the involvement of downstream transcription factors in denticle polarity. The transcription factor TCF , though mutant protein levels are high and junctions appear normalF1aarm (M/Z) embryos expressing Wg along the ventral midline, we observed some reorientation of denticles toward the midline (compare F1aarm mutantF1aarm (M/Z) mutants may be through the membrane pool of Arm at the adherens junctions. Taken together, our results suggest that proper PCP in the epidermis requires both a transcriptional response of Wg and Hh and thereby a functional Arm in the nucleus, as well as a functional Arm at adherens junctions.Finally, we investigated whether Arm is required for the transmission of the Wg polarity signal. We approached this by using an allelic series of  compare . This alDrosophila embryonic epidermis to establish planar cell polarity. Though the complete molecular mechanisms that control the complex system of PCP in the ventral epidermis remain to be determined, this process appears to occur in part through the asymmetric localization of Arm at the membrane. Further, proper polarity signaling is abolished if specific phosphorylation sites within the α-catenin binding domain of Arm are mutated. These sites were originally found to increase the affinity of β-catenin for α-catenin when phosphorylated by Casein Kinase II in vitro, suggesting one mechanism for stabilizing junctionsin vivo support for this hypothesis, as low levels of ArmAA rescued cellular junction defects to a similar extent as expression of an α-catenin/E-cadherin fusion protein, a protein that makes overly stable junctions AA expression lead to apparent polarity defects. As ArmAA does not localize asymmetrically the way that wild-type Arm does, we inferred that CKII phosphorylation may be required for the accumulation of junctions in specific regions of cells implying that stable junctions at specific sites in a cell are required for proper planar cell polarity. Further, our findings revealed that when all signaling activity is abolished through null mutations in the Wg or Hh signaling pathways, both cell identity and polarity determination was disrupted. It remains to be determined how Wg and Arm proteins function in polarity signaling, specifically whether they work through known PCP components, function similarly to their role in dorsal closure, or perhaps through novel signaling mechanisms like the interaction with Notch or AxinOur results indicate that Wg and Hh act as instructive cues in the wg and hh genes are required for the proper establishment of cell identities within segmentsThe hh is absent, suggesting that when the hh gene is present a boundary may be formed, thus preventing Wg from orienting the denticles to the same extent. It also appears that the distance over which Wg can act is longer in the absence of hh as expected from previous observationsIn the early embryo, expression of Wg and Hh is determined by pair-rule genes, but this effect is transient and requires mutually reinforcing positive activation loops to form between cells expressing Wg and En/HhC. elegans studies, Wnts have been implicated in the establishment of polarity, but only one study in Drosophila suggested a role for Wg in PCPtcf or ci mutations respectively prevents the polarizing activity of both pathways. This is similar to the PCP disruptions found in the Drosophila eye model for Wg signaling componentsF1Aarm mutant the orientation of denticles can be affected by the expression of Wg without affecting the cell-fates, suggesting that perhaps Wg can affect polarity directly. This effect of Wg was not observed in stronger arm mutant embryos suggesting that Arm protein is required for the Wg effect on denticle orientation. Interestingly, cell culture work has recently implicated Wg in controlling adherens junction strengthThe PCP signaling pathway determines planar polarity in a variety of tissuesarm function, and efficient ubiquitous or directional ectopic expression. Unfortunately, the major limitation of the ventral midline expression assay is that it only works for secreted, diffusible ligands. Thus, cell-autonomous activation of Hh or Wg pathway components (such as with activated Arm or Smo) along the ventral midline cannot be observed, since these cells invaginate and do not become a part of the external epidermis. We are currently working on ways to overcome this technical limitation.The use of the embryonic epidermis allowed us to observe the interesting possibility that Arm functions in cell polarity. Since some of the molecules involved in the PCP signaling pathway are similar to Cadherinsin vivo and in vitro have challenged thisThe fact that β-catenin is both an oncogene and a component of adherens junctions has led to many studies attempting to link the phosphorylation state of β-catenin in adherens junctions to the epithelial to mesenchymal transition (EMT) in cancer cells and during development. Phosphorylation of tyrosine residues in β-catenin is thought to lead to disassembly of adherens junctionsAA can rescue the basic activity of junctions lost in strong arm mutant embryos, which is to hold a tissue together. However, its reduced affinity for α-catenin does not cause a local increase in α-catenin levels and therefore Actin levels do not become asymmetric. This leads to a skewing of the normal polarization of the Actin cytoskeleton. It will be crucial to determine how junctions are localized asymmetrically in the first place, and whether this is dependent on extracellular signaling. These findings, and the effects of α-catenin mutations on inflammation and tumor progression in the mouse epidermisInterestingly, the recent findings that α-catenin and β-catenin do not form a stable complex in junctions These experiments provide some of the first evidence that the Hh signaling pathway is involved in polarity. It is particularly interesting that Hh expression leads to the reorganization of Actin structures within epithelial cells, since this suggests that Hh can affect the polarity of the Actin cytoskeleton. This finding is also relevant to cancer biology, because during metastasis, cancer cells lose polarity and essentially ignore their environment. Our results show that Wnts and Hh can affect cell polarity, in addition to their well-known effects on cell proliferationarm mutations O43A01arm, F1Aarm, XM19arm, and XM19arm, M11-1zw3 double mutant are described in detailCX4, pan2, ciCe2, hh3, smo2, ptcIN108wg and V26dsh see Flybase (http://flybase.bio.indiana.edu/).Generation of germline clone embryos from flies with Maternal and zygotic mutant eggs were generated by the dominant female sterile technique AA construct was generated with T111A and T121A mutations through site-directed mutagenesis with primers 5′-CCGGAAGCCCTGGAGGAGGGCATTGAGA TTCCCTCCGCCCAGTTTGAT and its complement (Quickchange from Stratagene), and fused to an N-terminal double HA tag in pUAStarm allele was wild-type. This results from the fact that only stabilized forms of Arm can overcome the degradation machinery and give phenotypes in the embryoThe ArmThe HA 3F10 rat antibody was from Roche. Anti Armadillo, Wingless, Patched, Sexlethal, Actin, and E-Cadherin antibodies were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Fixations, stainings, and cuticle preps as described previouslyF1Aarm (M/Z), and O43A01arm (M/Z) mutants expressing ArmAA, 300–600 denticles were counted in each embryo (n = 4/genotype). Denticles that were pointing anteriorly or posteriorly were classified as denticles that were pointing correctly, while denticles that were pointing up or down were classified as denticles that were pointing incorrectly. We counted any denticle that was able to be scored within the field of view. Any denticle whose polarity was obscured or not in focus was left unscored in all images.40× phase contrast images of cuticles were analyzed for denticle polarity by eye in Adobe Photoshop. Cuticles were oriented such that anterior was to the left and posterior was to the right. Denticles that appeared to within 45 degrees to the right or to the left of exactly anteriorly, posteriorly, up, and down were pseudo-colored red, green, blue, and orange respectively. The very tip of the denticle showing the characteristic hook of each denticle was a useful marker in deciding in which direction the denticle was pointing. For quantitation of wild-type,


The subcellular localization of proteins is critical to their biological roles. Moreover, whether a protein is membrane-bound, secreted, or intracellular affects the usefulness of, and the strategies for, using a protein as a diagnostic marker or a target for therapy. We employed a rapid and efficient experimental approach to classify thousands of human gene products as either “membrane-associated/secreted” (MS) or “cytosolic/nuclear” (CN). Using subcellular fractionation methods, we separated mRNAs associated with membranes from those associated with the soluble cytosolic fraction and analyzed these two pools by comparative hybridization to DNA microarrays. Analysis of 11 different human cell lines, representing lymphoid, myeloid, breast, ovarian, hepatic, colon, and prostate tissues, identified more than 5,000 previously uncharacterized MS and more than 6,400 putative CN genes at high confidence levels. The experimentally determined localizations correlated well with in silico predictions of signal peptides and transmembrane domains, but also significantly increased the number of human genes that could be cataloged as encoding either MS or CN proteins. Using gene expression data from a variety of primary human malignancies and normal tissues, we rationally identified hundreds of MS gene products that are significantly overexpressed in tumors compared to normal tissues and thus represent candidates for serum diagnostic tests or monoclonal antibody-based therapies. Finally, we used the catalog of CN gene products to generate sets of candidate markers of organ-specific tissue injury. The large-scale annotation of subcellular localization reported here will serve as a reference database and will aid in the rational design of diagnostic tests and molecular therapies for diverse diseases. An important goal of current biological research is annotation of human genes with relevant descriptors and properties. One critical property of interest to biologists and medical researchers is the subcellular localization of gene products, as this affects a protein's biological role and our ability to use it as a therapeutic target. This study used a microarray-based functional genomic method that allows rapid, large-scale identification of subcellular localization, enabling the authors to annotate the localization of thousands of previously uncharacterized human gene products. The authors then provide an example of how these data can be used by applying them to the search for tumor-specific markers. Using data from hundreds of DNA microarray profiles of global gene expression patterns in tumors and normal tissues, they identify candidate genes encoding membrane-associated and secreted proteins that are highly overexpressed in tumors and that might therefore be particularly good targets for diagnostic tests or molecular therapies. Diagnostic tests based on these markers could potentially enable cancers to be detected earlier than is currently possible, and molecular therapies targeting the products of these genes could have high specificity for the corresponding cancers. The subcellular localization of proteins critically affects their biological roles and functions. For example, sequence-specific DNA-binding proteins can only alter transcriptional activity if they are localized to the nucleus, and transmembrane (TM) receptors can only bind their soluble ligands if they are at least partially exposed to the extracellular environment. Categorization of proteins by subcellular localization is therefore one of the essential goals for functional annotation of the human genome.Proteins that are inserted into membranes or secreted are a particularly important class, as they include signal transduction receptors, transporters, channels, cell-to-cell signaling molecules, extracellular matrix components, and adhesion molecules. Surface and secreted proteins are also of special relevance to many areas of medicine. Plasma membrane proteins and secreted signaling proteins are candidate targets for monoclonal antibody-based therapies . There aA variety of empirical and computational approaches has been developed for identifying membrane and secreted proteins. Commonly employed experimental approaches include the signal-sequence trap ,5, the sWe set out to classify thousands of human genes as encoding either MS or CN proteins, using a previously described genome-scale method . BrieflyUsing this approach, we identified more than 5,000 putative MS and more than 6,400 putative CN genes (UniGene clusters). Our annotations agreed well with in silico methods for predicting localization, but also included thousands of genes for which such predictions were not available. One advantage of our approach was the ability to directly apply our categorizations to microarray gene expression data from hundreds of primary tumor and normal tissue samples that were generated separately using the same DNA microarrays. We were thus able to identify hundreds of genes that showed tumor-specific expression patterns and that are highly likely to encode MS proteins. These data provide a foundation for the development of targeted therapies and diagnostic tests for a wide variety of human malignancies.Based on previously reported microarray experiments , we chosThe thousands of genes represented on the microarrays for which the subcellular localizations of the protein products have previously been identified provided internal standards for assessing the success of each fractionation and for determining the confidence with which an uncharacterized gene could be classified as MS or CN. Genes encoding proteins inserted into cellular membranes or that are secreted were designated as the “MS reference set,” while genes encoding cytoplasmic or nuclear proteins were designated as the “CN reference set.” Limiting our search to the 24,365 UniGene clusters that were detectably expressed in at least three of the samples, we used empirical localization data reported in the Swiss-Prot  and LocuTo estimate the number of novel MS or CN genes that were identified by each additional fractionation, we calculated the total number of unique cDNA clones that were more than 85% enriched in the membrane or cytosolic fraction on at least one array. As show in 2 Cy5/Cy3 ratio as the metric for our subsequent analyses. Clones were ranked in descending order of the average log2 Cy5/Cy3 ratio, and a moving average approach was used to identify the local percentage of characterized MS/CN proteins at each end of this distribution. Since a subset of the UniGene clusters included on the arrays was represented by two or more elements, we removed all clusters with ambiguous localizations . As expected, relaxing the local percentage enrichment allowed annotation of an increasingly larger set of unknown clones, but at the expense of specificity  for a number of parameters for every clone across all arrays, including: the local percentage of characterized MS genes based on the moving average analysis (see above), the base 2 logarithm of the Cy5/Cy3 ratio, the ratio of intensity to local background for both Cy3 and Cy5, and the background-subtracted intensity for both Cy3 and Cy5. As a final parameter, we included the ratio of the sum of Cy5 background-corrected intensities to the sum of Cy3 background-corrected intensities across all arrays. To identify the best classification approach, receiver–operator curves were generated using each of these parameters  was classified by our analysis as MS, but the protein is cataloged as cytoplasmic by Gene Ontology. While no other experimental data suggesting MS localization for RBP5 have been reported, the closely related protein RBP4, the serum retinol carrier, is a documented secreted protein . This suA third class of CN genes encoding proteins reported to be cytoplasmic or nuclear, but whose mRNAs fractionated with membranes in our analysis, included genes encoding proteins that function at or near cellular membranes, but are not integral membrane components themselves. ACK1 and TXK, two nonmembrane spanning kinases, were examples of this class. The protein encoded by ACK1 is localized to clathrin-containing vesicles , and theAnother potential explanation for unexpected enrichment of some CN gene transcripts in the membrane fraction applies to genes whose transcripts are alternatively spliced, with one splice form encoding a CN protein, while another encodes an MS protein. This is the case for estrogen receptor 1 (ESR1), which is generally classified as encoding a CN protein, but which our method classified as MS. While the best characterized role for ESR1 is as a transcriptional activator in the nucleus, several reports provide evidence for an alternatively spliced variant that encodes a surface-exposed membrane-bound protein that can participate in signal transduction ,26. InteWe also investigated genes whose products were reported to be secreted or localized to membranes but whose transcripts were enriched in the CN fraction. Using GO-TermFinder  we foundAnother class of membrane or secreted proteins whose mRNA reproducibly fractionates with the cytosol was represented by the small inducible cytokine subfamily E, member 1 (SCYE1). This gene encodes a multifunctional polypeptide with both cytokine and tRNA-binding activities. Studies have shown that the tRNA-binding domain interacts with aminoacyl-tRNA synthetases, while a different domain of the protein, with inflammatory cytokine activity, is released extracellularly by proteolysis in response to apoptotic stimuli . These eA particularly striking result of these studies was that 44% of the genes that we could classify were predicted from our results to encode MS proteins . PreviouThis allowed us to estimate the number of genes encoding MS proteins we would have expected to find had we been able to identify every one of them. In order to assess the robustness of our estimate, we performed the calculation with two MS gene sets, one derived from lower and the other from higher stringency analyses of our localization data. Using both sets, we estimated the total number of MS UniGene clusters present on our arrays to be approximately 7,000. Given that our arrays provided technically adequate data for 24,365 clusters, and assuming that these were representative of all genes in the genome, our data suggest that approximately 30% of human genes encode MS proteins. Thus, our empirical results are consistent with the upper extreme of the previous computational predictions. This analysis implies that there are likely to be thousands of still unrecognized surface-exposed and secreted proteins that could serve as useful diagnostic markers or therapeutic targets.http://microarray-pubs.stanford.edu/mbp2). We removed from this list all of the known genes encoding CN proteins that we had curated earlier. We next added any gene encoding a membrane or secreted protein that was identified by our previous database searches but was not identified as such in our analysis. This aggregate list contained approximately 7,300 putative genes (UniGene clusters), represented on our microarrays by 12,030 cDNA clones , AFP, EGFR, ERBB2, CEACAM5 (CEA), and VEGF. Their expression patterns were generally consistent with previous studies. For example, ERBB2 was especially highly expressed in a subset of breast tumors, while CEACAM5 was most highly expressed in several epithelial tumors, including those of the breast, stomach, and lung [and lung –53.http://microarray-pubs.stanford.edu/mbp2). This information can be used to help prioritize genes for follow-up studies.We next wished to identify potential therapeutic and diagnostic targets that were expressed in a tumor-specific fashion. Small molecule or monoclonal antibody-based therapies have shown promise as single agents or in conjunction with traditional modalities such as chemotherapy and radiotherapy, and it is likely that even better tumor control and cure rates could be achieved by developing combinations of biologically based drugs for each tumor type. The ideal class of markers for this approach consists of surface-exposed and secreted proteins that are highly expressed in tumor cells and only minimally expressed in normal tissues. The combination of our MS gene list and the large-scale gene expression dataset we constructed allowed us to rationally identify such candidate genes. We ranked genes based on the difference between the median expression in tumor samples of a given class and the 95th percentile expression level across all normal tissue samples. This resulted in selection of genes that were more highly expressed in most of the tumor samples than in the vast majority of normal tissues. To further prioritize candidate genes, we also incorporated an estimate of transcript abundance into our selection scheme, hypothesizing that more highly expressed genes will make better therapeutic or diagnostic targets. Since all of the microarray data presented here were generated using a two-color comparative hybridization approach that produces measurements of relative abundance between different samples, we aimed to identify the relative transcript abundance of potential candidate markers within each tumor class. To estimate transcript abundance, we used data from comparative hybridizations of the common reference RNA used in each tumor experiment versus normal female genomic DNA . These dERBB2 for ERBB2-positive breast cancer [VEGF for renal cell carcinoma [MS4A1 (CD20) for non-Hodgkin's lymphoma [EGFR was the top gene selected for glioblastoma, where this gene has been shown to be the most frequently amplified protooncogene [http://microarray-pubs.stanford.edu/mbp2). cDNA elements representing ERBB2 made up three of the top five elements selected for the ERBB2 breast cancer subtype, but none of these were included in the lists for basal and luminal breast cancers. Conversely, CDH3, one of the known basal breast cancer markers, was only identified as a strong marker for basal breast tumors. These findings suggest that overlapping but nonidentical cocktails of molecularly targeted drugs may allow the best distinction between tumor and normal tissues for various tumor subtypes. Among genes not previously well known as tumor markers were several genes with expression patterns highly specific for more than one tumor compared to all normal tissues. These included the receptor tyrosine kinase AXL, which was overexpressed in luminal breast, testicular, and ovarian cancers. Genes such as AXL may represent particularly fruitful candidates for biologically based therapies, as they could potentially have activity against a number of different tumors.As shown in t cancer , VEGF foarcinoma , and MS4lymphoma . A cDNA oncogene . For braoncogene ,42. Overt-test to identify the 20 genes most consistently expressed at a higher level in each of the tissues compared to all others , represented on our DNA microarrays by 15,311 cDNA clones, using an approach analogous to that used to assemble the aggregate list of putative MS proteins in the previous section see . Focusinl others . We founpithelia , a patteia [KRT5 . AntibodWe have used DNA microarrays coupled with subcellular fractionation of mRNAs to categorize thousands of genes as either MS or CN. Our classifications agreed well with in silico methods for predicting subcellular localization. The fact that approximately 40% of all previously identified MS proteins, and a similar fraction of MS proteins identified in this work, do not contain predicted SPs or TM domains underscores the continuing importance of experimental approaches to determining the subcellular localization of proteins. It is important to note that both the computational methods and our microarray approach provide only indirect evidence for a specific localization, ultimately requiring direct validation to conclusively determine a protein's localization. Annotation of genes based on both experimental data and in silico predictions will provide the surest assignments. Based on our data, we estimate that approximately 30% of human genes encode MS proteins, a proportion at the upper end of previous estimates . This esWe identified a subset of genes encoding known MS or CN proteins whose mRNAs fractionated in an unexpected manner. While one common cause of this phenomenon was incorrect or incomplete annotation of the localization of the proteins encoded by these genes, we also identified intriguing examples that reflected important aspects of the molecular regulation and function of this class of genes. The presence of genes encoding cytoplasmic proteins that function near membrane structures in the group of CN-encoding transcripts with unexpected membrane localization suggests that cells actively sort mRNAs in order to promote their translation at sites where their encoded protein products are required. A recent study in yeast showed that some RNA-binding proteins specifically associate with mRNAs encoding membrane-associated proteins , and a sThe microarray-based approach we used to study subcellular localizations of mRNAs can still be further developed and refined. Simply fractionating more cell lines and extending the approach to whole tissues should allow the classification of many more genes. Technically, it may be possible to refine the specificity of the method by analyzing only those transcripts in the MS and CN fractions that are also associated with polyribosomes. Similarly, approaches identifying mRNAs associated with subcellular organelles, cytoskeletal elements, motor proteins, and RNA-binding proteins will further our understanding of subcellular localization of RNAs and uncover interesting aspects of the biology of the proteins that they encode ,60–63. MUsing publicly available gene expression data, we identified hundreds of MS genes with tumor-specific expression patterns. The proteins encoded by these genes represent promising targets for antibody- or small molecule-based therapeutics, for blood based assays for early detection of cancer, for monitoring of treatment responses, and for detecting recurrence following treatment. While there was prior evidence for tumor-specific expression of some of the markers, we identified many novel tumor markers that expand the catalog of potentially useful drug targets. Our approach also allows rational prioritization of known and novel markers for drug development based on relative specificity of expression and absolute transcript abundance, increasing the likelihood of developing successful therapies. Our strategy could be refined by developing methods to focus separately on surface and secreted proteins, as these may be better candidates for therapeutic and diagnostic markers, respectively. Also, expansion of the number of tumor and normal samples with large-scale gene expression data would allow even better accuracy in selecting tumor-specific markers. Our approach might also be extended to anticipate and avoid molecular targets that would risk potential clinical side effects, such as excluding genes highly expressed by hematologic cells to limit bone marrow toxicity or by excluding genes expressed by cardiac myocytes in order to minimize cardiovascular toxicity. The combination of functional annotations such as subcellular localization with systematic data on gene expression in the gamut of normal cells and tissues as well as cancers provides a basis for improved approaches to drug design and diagnosis.The methods closely followed those of a previous study . Brieflyhttp://cmgm.stanford.edu/pbrown). To quantitate the distribution of mRNAs between the membrane and cytoplasmic fractions, Cy5-labeled cDNA was prepared from RNA extracted from the rough ER fractions, and Cy3-labeled cDNA was prepared from RNA extracted from the cytoplasmic complement. The cDNA microarrays were produced by the Stanford Functional Genomics Facility and contained a set of approximately 42,000 sequence-confirmed cDNA clones, representing both characterized and uncharacterized genes. Raw images and data from the experiments described here are available at http://microarray-pubs.stanford.edu/mbp2 and the Stanford Microarray Database [DNA microarrays were produced and hybridized as previously described  .Analyses were performed as described in the text and figure legends. Perl scripts were used where necessary to facilitate the analyses. For in silico predictions of localization, we used the SignalP program (HMM/Smean score method)  for SPs Dataset S1Experimental, in silico, and known localization data for each well-measured clone.(7.2 MB XLS)Click here for additional data file.Dataset S2List of 12,030 MS clones used for the analyses in (156 KB TXT)Click here for additional data file.Dataset S3List of 15,311 CN clones used for the analyses in (196 KB TXT)Click here for additional data file.Figure S1In order to examine the relationship between tumor and normal specimens stemming from the same tissue, mean centroids were calculated for each tumor and normal tissue group. These were then hierarchically clustered using average linkage clustering. Tumor and normal classes from the same tissue generally clustered together on terminal branches, with a few exceptions .(868 KB EPS)Click here for additional data file.Protocol S1(60 KB DOC)Click here for additional data file.Table S1The table lists the tissue of origin for each line and its ATTC catalog number (where available).(30 KB DOC)Click here for additional data file.Table S2p-values were calculated using GO-TermFinder.Overrepresentation of GO annotations among characterized MS genes, whose transcripts were enriched in the cytosolic fraction compared to all characterized MS genes, is shown. Corrected (48 KB DOC)Click here for additional data file.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) accession numbers for the genes and proteins discussed in this paper are AFP (174), AXL (558), CDH3 (1001), CEACAM5 (1048), cyclin B1 (891), cyclin B3 (85417), cyclin E2 (9134), EGFR (1956), ERBB2 (2064), ESR1 (2099), GNAS (2778), GOSR2 (9570), KLK3 (354), KRT5 (3852), MS4A1 (2206), PGR (5241), PTCH (5727), RBP4 (5950), RBP5 (83758), SCYE1 (9255), secretagogin (10590), SEC61B (10952), stomatin (2040), STX17 (55014), TFAP2C (7022), and VEGF (7422).The Entrez Gene (


Cardiac resynchronisation therapy (CRT) using biventricular (BIV) pacing has proved its effectiveness to correct myocardial asynchrony and improve clinical status of patients with severe congestive heart failure (CHF) and widened QRS. Despite a different effect on left ventricular electrical dispersion, left univentricular (LV) pacing is able to achieve the same mechanical synchronisation as BIV pacing in experimental studies and in humans. This results in clinical benefits of LV pacing at mid-term follow-up, with significant improvement in functional class, quality of life and exercise tolerance at the same extent as those observed with BIV stimulation in non randomised studies. Furthermore these benefits are obtained at lesser costs and with conventional dual-chamber devices. However, LV pacing has to be compared to BIV pacing in randomised trials before being definitely considered as a cost-effective alternative to BIV pacing. Despite advances in drug treatment, congestive heart failure (CHF) remains a major health care problem associated with a poor quality of life and a high mortality rate. During the past decade, cardiac resynchronisation therapy (CRT) using biventricular (BIV) pacing emerged as a promising technique improving quality of life, exercise tolerance and mortality -3, and iMany other arguments support the concept that LV univentricular pacing by reversing intraventricular dysynchrony is sufficient in humans to improve LV function and clinical status at the same extent and at a lower cost-effective ratio than BIV pacing and this is in accordance with the fact that intraventricular asynchrony seems more relevant that interventricular asynchrony to predict prognosis in patients with dilated cardiomyopathy .The first clinical series with CRT were performed empirically using BIV pacing . However2 (2.46 ml/mn/kg), in 6 minute walk distance (47 m) and a decrease in quality of life score (8.1) during the active pacing period in the group of patients with the longest  QRS duration (above 150 ms) [2 by 26 %) and norepinephrine level by 37% [Whether or not these hemodynamic benefits result in sustained improvement of clinical status during permanent LV stimulation has not been extensively investigated until now. However, some studies reported significant improvement in both exercise tolerance and quality of life at mid-term  follow-up during chronic LV pacing ,19 or fa 150 ms) . In anot 150 ms) . Our perl by 37% .Are these clinical effects correlated with improvement in myocardial synchrony? This is an important issue on a physiopathological point of view and for the selection of candidates for LV pacing. In an interesting experimental study using a canine model of cardiac failure and left bundle branch block, Leclercq et al examined mechanical and electrical synchrony during right atrial, LV and BIV stimulation; as expected, electrical dispersion decreased during BIV pacing whereas it increased during LV pacing; however, despite this opposite action upon electrical phenomena, the same improvement in mechanical coordination within the LV, measured by tagged magnetic resonance imaging, was observed with LV and BIV pacing, and this was correlated with improvement in hemodynamics (25 % increase in dP/dt and aortic pulse pressure) . SimilarBy comparison to BIV pacing, LV pacing offers several technical and economical advantages : 1) if insertion of a third lead (the right ventricular one) is not the most difficult part of the operative procedure, it could prolong significantly its duration and the X-ray exposure; 2) the presence of 3 leads instead  of 2 induces certainly more adverse events and increases the risk of venous thrombosis or mechanical complications; 3) more importantly, uniLV pacing needs only a less complex to program and less often subject to dysfunction conventional dual-chamber  pacemaker. Finally, BIV pacing is more expensive than LV pacing as it implies implantation of a supplementary right ventricular lead and of a specific and more costly device: in Europe, the extra cost could be approximately evaluated at 30 % and this must be taken in account in the context of the growing “epidemic” of heart  failure consecutive to demographic ageing and of the control of medical expenses in many countries.So, at this time, there are no arguments in favour of the hypothesis that, for patients with severe CHF and enlarged QRS duration, BIV pacing would be significantly more effective than LV pacing in terms of mechanical coordination, hemodynamic and clinical benefits. However, our knowledge about the effects of LV pacing still suffers some limitations: the present studies are only observational and non-randomised. Furthermore, it seems, as shown during acute hemodynamic studies, that in some patients LV pacing would not be as effective as BIV pacing , and this could perhaps be the case in patients with a dysfunctional right venricle but no study have focused on this topic at this time. Finally  and unlike BIV pacing ,23, no s


WRN gene product that possesses ATPase, 3' to 5' helicase and 3' to 5' exonuclease activities. Although WRN has been most prominently suggested to function in telomere maintenance, resolution of replication blockage and/or recombinational repair, its exact role in DNA metabolism remains unclear. WRN is the only human RecQ family member to possess both helicase and exonuclease activity, but the mechanistic relationship between these activities is unknown. In this study, model single-stranded and 3' overhang DNA substrates of varying length and structure were used to examine potential coordination between the ATPase/helicase and exonuclease activities of WRN.The cancer-prone and accelerated aging disease Werner syndrome is caused by loss of function of the Our results show that WRN can not only bind to but also catalyze the 3' to 5' degradation of single-stranded and 3' overhang DNA substrates, structures that were previously thought to be refractory to WRN exonuclease activity. The length of the single-stranded regions in these structures is a critical parameter in determining both the binding affinity and the level of exonuclease activity of WRN. Most importantly, specific nucleotide cofactors dramatically stimulate WRN exonuclease activity on these substrates, with conditions that permit ATP hydrolysis not only resulting in enhanced exonuclease activity but also altering its length dependence on these structures. Parallel experiments show that a deletion mutant containing only the WRN exonuclease domain lacks both this DNA length and nucleotide cofactor dependence, demonstrating that the interaction of the ATPase/helicase domain of WRN with the DNA substrate has a profound influence on exonuclease activity.Our results indicate that, under conditions that permit ATP hydrolysis, there is a dynamic and cooperative relationship between the distinct ATPase/helicase and exonuclease domains of WRN with regard to their orientation on DNA. Based on these results, models are proposed for the coordinated, unidirectional 3' to 5' movement of the helicase and exonuclease domains of WRN on DNA that should be informative for elucidating its function in genome maintenance. Werner syndrome (WS) is a rare autosomal recessive disease displaying accelerated development of many characteristics associated with normal aging including graying and loss of hair, cataracts, osteoporosis, atherosclerosis, hypertension and increased frequencies of diabetes mellitus type II and specific types of cancer (reviewed in -6). IndiWRN belongs to the RecQ family of helicases  that hasThe WRN protein possesses a central ATPase/helicase domain composed of defined sequence motifs that are highly homologous within RecQ family members. In all RecQ helicases characterized thus far, this domain is essential for both (single-stranded) DNA-dependent ATPase and nucleic acid unwinding activities ,12. WRN in vitro studies, the catalytic functions of the exonuclease and helicase domains can be isolated by making either key amino acid substitutions in each domain or deletion mutants lacking one region or the other triphosphate, EMSA: electrophoretic mobility shift assays, NP-40: Nonidet P-40, PAGE: polyacrylamide gel electrophoresis, PNK: T4 polynucleotide kinase, SDS: sodium dodecyl sulfate, WS: Werner syndromeAM participated in the design of the experiments and performed specific biochemical assays, prepared materials for publication, and helped draft the manuscript. XL carried out biochemical assays and prepared DNA substrates and purified proteins. DKO conceived of the study, oversaw the design of all experiments, and participated in drafting the manuscript. Each author has read and approved the final version of the manuscript.Specificity of WRN exonuclease activity on a 3' overhang substrate Data showing that the exonuclelease activity observed on 3' overhang substrates is inherent to WRN protein.Click here for fileSilver stain analysis of purified recombinant WRN protein Purified WRN analyzed by SDS-PAGE followed by silver staining.Click here for file