Progressive changes in adherens junction structure during intestinal adenoma formation in Apc mutant mice

The C57BL/6J-Min/+ (Min/+) mouse bears a mutant Apc gene and therefore is an important in vivo model of intestinal tumorigenesis. Min/+ mice develop adenomas that exhibit loss of the wild-type Apc allele (Apc(Min/-)). Previously, we found that histologically normal enterocytes bearing a truncated Apc protein (Apc(Min/+)) migrated more slowly in vivo than enterocytes with either wild-type Apc (Apc(+/+)) or with heterozygous loss of Apc protein (Apc(1638N)). To study this phenotype further, we determined the effect of the Apc(Min) mutation upon cell-cell adhesion by examining the components of the adherens junction (AJ). We observed a reduced association between E-cadherin and beta-catenin in Apc(Min/+) enterocytes. Subcellular fractionation of proteins from Apc(+/+), Apc(Min/+), and Apc(Min/-) intestinal tissues revealed a cytoplasmic localization of intact E-cadherin only in Apc(Min/+), suggesting E-cadherin internalization in these enterocytes. beta-Catenin tyrosine phosphorylation was also increased in Apc(Min/+) enterocytes, consistent with its dissociation from E-cadherin. Furthermore, Apc(Min/+) enterocytes showed a decreased association between beta-catenin and receptor protein-tyrosine phosphatase beta/zeta (RPTPbeta/zeta), and Apc(Min/-) cells demonstrated an association between beta-catenin and receptor protein-tyrosine phosphatase gamma. In contrast to the Apc(Min/+) enterocytes, Apc(Min/-) adenomas displayed increased expression and association of E-cadherin, beta-catenin, and alpha-catenin relative to Apc(+/+) controls. These data show that Apc plays a role in regulating adherens junction structure and function in the intestine. In addition, discovery of these effects in initiated but histologically normal tissue (Apc(Min/+)) defines a pre-adenoma stage of tumorigenesis in the intestinal mucosa.     

One dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induces tumours in Min/+ mice by truncation mutations or LOH in the Apc gene

The C57BL/6J-Min/+ (multiple intestinal neoplasia) mouse has a heterozygous nonsense Apc(Min) (adenomatous polyposis coli) mutation, and numerous adenomas spontaneously develop in the intestine. Neonatal exposure of Min/+ mice to the food carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) or 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) (one injection of 50mg/kg) increased the number of small intestinal tumours about three- and two-fold, respectively. The number of colonic tumours was only increased in males. We examined whether the wild-type Apc allele was affected in intestinal tumours induced by either PhIP or IQ. In spontaneously formed and in IQ-induced small intestinal and colonic tumours from these mice, the main mechanism for tumour induction was loss of wild-type Apc allele, i.e. loss of heterozygosity (LOH). In contrast to the IQ-induced (84% LOH) and spontaneously (88% LOH) formed tumours, only 55% of the PhIP-induced small intestinal tumours from males showed LOH. Tumours that apparently had retained the wild-type Apc allele were further analysed for the presence of truncated Apc proteins by the in vitro synthesised protein (IVSP) assay. Truncated Apc proteins, indicating truncation mutations in exon 15 of the Apc gene, were detected in two of the 12 PhIP-induced tumours in segment 2 (codons 686-1217), and two of five IQ-induced tumours, one in segment 2 and the other in segment 3 (codons 1099-1693). Three of these four mutations, all in segment 2 of the Apc gene, were confirmed by sequencing. The PhIP-induced mutations were detected at codon 1125 (C deletion) and 1130 (G-T transversion), and the IQ-induced mutation was at codon 956 (C-T transition). Importantly, no truncated proteins were detected in tumours from unexposed mice with apparently retained wild-type Apc allele. These results show that one injection of either PhIP or IQ induces intestinal tumours in the Min/+ mice by inactivation of the wild-type Apc allele either by causing LOH or truncation mutations.     

Interleukin-6 and cachexia in ApcMin/+ mice

The Apc(Min/+) mouse has a mutation in the Apc tumor suppressor gene and develops intestinal polyps, beginning at 4 wk of age. This mouse develops cachexia by 6 mo, characterized by significant loss of muscle and fat tissue. The purpose of the present study was to determine the role of circulating interleukin-6 (IL-6) and the polyp burden for the development of cachexia in Apc(Min/+) mice. At 26 wk of age, mice exhibiting severe cachectic symptoms had a 61% decrease in gastrocnemius muscle weight, complete loss of epididymal fat, a 10-fold increase in circulating IL-6 levels, and an 89% increase in intestinal polyps compared with mildly cachectic animals. Apc(Min/+)/IL-6(-/-) mice did not lose gastrocnemius muscle mass or epididymal fat pad mass while overall polyp number decreased by 32% compared with Apc(Min/+) mice. Plasmid-based IL-6 overexpression in Apc(Min/+)/IL-6(-/-) mice led to a decrease in gastrocnemius muscle mass and epididymal fat pad mass and increased intestinal polyp burden. IL-6 overexpression did not induce cachexia in non-tumor-bearing mice. These data demonstrate that IL-6 is necessary for the onset of adipose and skeletal muscle wasting in the Apc(Min/+) mouse and that circulating IL-6 can regulate Apc(Min/+) mouse tumor burden.     

Analysis of reciprocal congenic lines reveals the C3H/HeJ genome to be highly resistant to ApcMin intestinal tumorigenesis

Genetic background affects polyp development in the Multiple intestinal neoplasia (Apc(Min)) mouse model. The Modifier of Min 1 (Mom1) locus accounts for approximately 50% of the variation in polyp multiplicity. We generated reciprocal congenic lines, such that the recipient C57BL/6J (B6) strain carries a donor C3H/HeJ (C3H) Mom1 allele, and the recipient C3H strain carries a donor B6 Mom1 allele. Hybrid progeny from congenic females mated to B6 Apc(Min/+) males were analyzed. A single C3H Mom1 locus on the B6 background reduced small intestinal polyp numbers by 50% and colon polyp incidence by 66% compared to their susceptible B6 Mom1(S/S)Apc(Min/+) siblings. These findings show that the C3H genome contains a resistant Mom1(R) locus. The reciprocal congenic line, which carries the susceptible B6 Mom1(S) locus on the C3H background, reduced small intestinal polyp numbers by 80% and colon polyp incidence by 95% compared to B6 Mom1(S/S)Apc(Min/+) mice. These data demonstrate that unidentified modifiers in the C3H strain can suppress intestinal polyp multiplicity in Apc(Min/+) mice, and act in the absence of a resistant Mom1(R) locus.     

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces genetic changes in murine intestinal tumours and cells with ApcMin mutation

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the mutagenic heterocyclic amines derived from cooked meat. In previous animal studies, spontaneous tumour formation in B6(Min/+) mice was associated with somatic loss of the wild-type Apc+ allele by loss of the entire chromosome 18 or by recombination. The objective of this study was to examine genetic changes caused by PhIP-exposure in a mouse intestinal cell line and in tumours from hybrid mice by keeping track of the chromosomes carrying the two Apc alleles. We transformed the SV40 T-immortalised intestinal epithelial cell line IMCE, derived from the B6(Min/+) mice by exposure to N-OH-PhIP, and studied the effect on Apc status and chromosome 18. Eighteen transformed cultures were obtained and all of them had retained the Apc+ allele. Five of seven transformed cultures were tumorigenic after implantation in nude mice. Chromosomal analysis of these five cultures and the parent IMCE cell line showed that the IMCE cells were near-tetraploid with an average of 77 chromosomes/cell, while the tumorigenic cell cultures were all triploid to hyper-triploid with a range of 61-69 chromosomes/cell. The number of copies of chromosome 18 was about four in the IMCE line and this copy number was retained in the transformed lines derived from IMCE. Changes in chromosome 18 and Apc during tumour development in vivo were examined in spontaneously formed and PhIP-induced intestinal tumours from two hybrid mice strains, i.e. B6(Min/+) - a murine FAP model - crossed with either AKR/J or A/J. We evaluated the allelic status of Apc, and the heterogenic microsatellite markers D18Mit19 and D18Mit4, located at the upper and lower ends of chromosome 18, respectively. In tumours from untreated animals, instability in the D18Mit19 and Apc was observed. Upon PhIP exposure, the B6(Min/A+) hybrid mouse tumours differed distinctly in genetic profile from those obtained from untreated animals and we detected three genetically different tumour groups, all of which had apparently retained Apc+. One group had allelic balance between the Apc(Min) and Apc+, the second had allelic imbalance between the Apc and D18Mit4 alleles, indicative of chromosomal stability in the first group and instability in the lower end of chromosome 18 in the second group, respectively. The third group showed variable allelic status of the three markers. A similar change in genetic profile was also seen in intestinal tumours of PhIP-exposed B6(Min/AKR+) hybrid mice, but it was less pronounced. Chromosomal breaks and/or recombinational events could be alternative explanations for the observed allelic imbalances in chromosome 18 markers in intestinal tumours from PhIP-exposed mice.     

Adenomatous polyposis coli truncation mutations in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced intestinal tumours of multiple intestinal neoplasia mice

The heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induces intestinal tumours in C57BL/6J-multiple intestinal neoplasia (Min)/+ mice. The main mechanism for PhIP-induced tumour induction in Min/+ mice is loss of the wild-type adenomatous polyposis coli (Apc) allele, i.e. loss of heterozygosity (LOH). In this study, single injections of either 10, 17.5 or 25 mg/kg PhIP on days 3-6 after birth all increased the mean number of small intestinal tumours two to three-fold, from 37.7 in controls to 124.8 in the PhIP-treated Min/+ mice. In total, we analysed 292 small intestinal tumours and 253 of these had LOH. The frequency of LOH in the Apc gene was 88, 93, 83 and 84% in tumours of 0, 10, 17.5 and 25 mg/kg PhIP-treated mice, respectively. Therefore, these lower doses of PhIP did not reduce the frequency of LOH, as found in our previous study with a single injection of 50 mg/kg PhIP (Mutat. Res. 1-2 (2002) 157). In the second part of this study, we wanted to characterise Apc truncation mutations from tumour samples apparently retaining the Apc wild-type allele from this and two previous experiments with PhIP-exposed Min/+ mice. In the first half of exon 15 in Apc, we verified 25 mutations from 804 tumour samples of PhIP-treated mice. Of these were 60% G-->T transversions, and 16% G deletions, indicating that these are the predominant types of PhIP-induced truncation mutations in the Apc gene in Min/+ mice. Most of the mutations were located between codon 989 and 1156 corresponding to the first part of the beta-catenin binding region. We also identified two Apc truncation mutations from 606 spontaneously formed intestinal tumours from untreated Min/+ mice, one C-->T transition and one T insertion, which were different from those induced by PhIP.     

Direct tandem mass spectrometry reveals limitations in protein profiling experiments for plasma biomarker discovery

The low molecular weight plasma proteome and its biological relevance are not well defined; therefore, experiments were conducted to directly sequence and identify peptides observed in plasma and serum protein profiles. Protein fractionation, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) profiling, and liquid-chromatography coupled to MALDI tandem mass spectrometry (MS/MS) sequencing were used to analyze the low molecular weight proteome of heparinized plasma. Four fractionation techniques using functionally derivatized 96-well plates were used to extract peptides from plasma. Tandem TOF was successful for identifying peptides up to m/z 5500 with no prior knowledge of the sequence and was also used to verify the sequence assignments for larger ion signals. The peptides (n>250) sequenced in these profiles came from a surprisingly small number of proteins (n approximately 20), which were all common to plasma, including fibrinogen, complement components, antiproteases, and carrier proteins. The cleavage patterns were consistent with those of known plasma proteases, including initial cleavages by thrombin, plasmin and complement proteins, followed by aminopeptidase and carboxypeptidase activity. On the basis of these data, we discuss limitations in biomarker discovery in the low molecular weight plasma or serum proteome using crude fractionation coupled to MALDI-MS profiling.     

Intracellular role for sphingosine kinase 1 in intestinal adenoma cell proliferation

Sphingosine kinase (Sphk) enzymes are important in intracellular sphingolipid metabolism as well as in the biosynthesis of sphingosine 1-phosphate (S1P), an extracellular lipid mediator. Here, we show that Sphk1 is expressed and is required for small intestinal tumor cell proliferation in Apc Min/+ mice. Adenoma size but not incidence was dramatically reduced in Apc Min/+ Sphk(-/-) mice. Concomitantly, epithelial cell proliferation in the polyps was significantly attenuated, suggesting that Sphk1 regulates adenoma progression. Although the S1P receptors (S1P1R, S1P2R, and S1P3R) are expressed, polyp incidence or size was unaltered in Apc Min/+ S1p2r(-/-), Apc Min/+ S1p3r(-/-), and Apc Min/+ S1p1r(+/-) bigenic mice. These data suggest that extracellular S1P signaling via its receptors is not involved in adenoma cell proliferation. Interestingly, tissue sphingosine content was elevated in the adenomas of Apc Min/+ Sphk1(-/-) mice, whereas S1P levels were not significantly altered. Concomitantly, epithelial cell proliferation and the expression of the G1/S cell cycle regulator CDK4 and c-myc were diminished in the polyps of Apc Min/+ Sphk1(-/-) mice. In rat intestinal epithelial (RIE) cells in vitro, Sphk1 overexpression enhanced cell cycle traverse at the G1/S boundary. In addition, RIE cells treated with sphingosine but not C6-ceramide exhibited reduced cell proliferation, reduced retinoblastoma protein phosphorylation, and cyclin-dependent kinase 4 (Cdk4) expression. Our findings suggest that Sphk1 plays a critical role in intestinal tumor cell proliferation and that inhibitors of Sphk1 may be useful in the control of intestinal cancer.     

KYSS: Mass spectrometry data quality assessment for protein analysis and large-scale proteomics

We introduce the computer tool “Know Your Samples” (KYSS) for assessment and visualisation of large scale proteomics datasets, obtained by mass spectrometry (MS) experiments. KYSS facilitates the evaluation of sample preparation protocols, LC peptide separation, and MS and MS/MS performance by monitoring the number of missed cleavages, precursor ion charge states, number of protein identifications and peptide mass error in experiments. KYSS generates several different protein profiles based on protein abundances, and allows for comparative analysis of multiple experiments. KYSS was adapted for blood plasma proteomics and provides concentrations of identified plasma proteins. We demonstrate the utility of the KYSS tool for MS based proteome analysis of blood plasma and for assessment of hydrogel particles for depletion of abundant proteins in plasma. The KYSS software is open source and is freely available at http://kyssproject.github.io/.     

热蛋白质组学分析：一种全面评估蛋白质状态的技术

热蛋白质组学分析（thermal proteome profiling,TPP）是细胞热漂移测定（cellular thermal shift assay,CETSA）与定量质谱（quantitative mass spectrometry,MS）的结合,所以也称为MS-CETSA。热蛋白质组学分析通过测量不同加热温度下细胞或细胞裂解物中可溶蛋白的含量来确定整个蛋白质组的稳定性。蛋白质可以在与药物或代谢物等小分子、核酸或其他蛋白质相互作用或在翻译后修饰时改变其热稳定性,而热蛋白质组学分析可以根据有无配体结合蛋白质的热稳定性差异来确定靶蛋白。目前热蛋白质组学分析已成功应用于识别药物的靶点和脱靶点,探究蛋白质-代谢物和蛋白质-蛋白质的相互作用。总体上,国内对这个技术的了解仍然欠缺,对此,文中对热蛋白质组学分析的原理、方法、应用以及优势与局限性进行了综述。     

Decreased intestinal polyp multiplicity is related to exercise mode and gender in ApcMin/+ mice.

Moderate-intensity treadmill running can alter male Apc(Min/+) mouse polyp formation. This purpose of this study was to examine whether exercise mode differentially affects Apc(Min/+) mouse intestinal polyp development in male and female mice. Male and female Apc(Min/+) mice were randomly assigned to control, treadmill (18 m/min; 60 min/day; 6 days/wk), or voluntary wheel running (24-h access) groups. Nine weeks of training decreased total intestinal polyps by 29% in male treadmill runners (66 +/- 9; P = 0.038) compared with male controls (93 +/- 7). The number of large polyps (>/=1-mm diameter) were also reduced by 38% in male treadmill runners (49 +/- 6; P = 0.005) compared with male controls (79 +/- 6). Treadmill running in female Apc(Min/+) mice and wheel running in both genders did not affect polyp number or size. Spleen weight decreased in male treadmill runners (91 +/- 9 mg; P = 0.011) and wheel runners (75 +/- 6 mg; P = 0.004) compared with controls (141 +/- 13 mg). Plasma IL-6 was reduced by 96% in male treadmill runners (1.2 +/- 0.6 pg/ml) and 78% in male wheel runners (6.6 +/- 3.3 pg/ml) compared with control mice (27.9 +/- 2.8 pg/ml; P < 0.05). Female mice responded similarly with an 86% decrease in plasma IL-6 with treadmill running (3.2 +/- 1.2 pg/ml) and 90% decrease with wheel running (2.9 +/- 2.0 pg/ml) compared with control mice (21.1 +/- 5.3 pg/ml; P < 0.05). The crypt depth-to-villus height ratio in the intestine, an indirect marker of intestinal inflammation, decreased by 21 (P = 0.024) and 24% (P = 0.029), respectively, in male and female treadmill runners but not wheel runners. Physical activity-induced attenuation of intestinal polyp number and size is dependent on exercise mode and differs between genders. The modulation of systemic and intestinal inflammation may also depend on exercise mode.     

Inhibition of intestinal polyposis with reduced angiogenesis in ApcMin/+ mice due to decreases in c-Myc expression

The c-myc oncogene plays an important role in tumorigenesis and is frequently deregulated in many human cancers, including gastrointestinal cancers. In humans, mutations of the adenomatous polyposis coli (Apc) tumor suppressor gene occur in most colorectal cancers. Mutation of Apc leads to stabilization of beta-catenin and increases in beta-catenin target gene expression (c-myc and cyclin D1), whose precise functional significance has not been examined using genetic approaches. Apc(Min/+) mice are a model of familial adenomatous polyposis and are heterozygous for an Apc truncation mutation. We have developed a model for examining the role of c-Myc in Apc-mediated tumorigenesis. We crossed c-myc(+/-) mice to Apc(Min/+) to generate Apc(Min/+) c-myc(+/-) animals. The compound Apc(Min/+) c-myc(+/-) mice were used to evaluate the effect of c-myc haploinsufficiency on the Apc(Min/+) phenotype. We observed a significant reduction in tumor numbers in the small intestine of Apc(Min/+) c-myc(+/-) mice compared with control Apc(Min/+) c-myc(+/+) mice. In addition, we observed one to three polyps per colon in Apc(Min/+) c-myc(+/+) mice, whereas only two lesions were observed in the colons of Apc(Min/+) mice that were haploinsufficient for c-myc. Moreover, reduction in c-myc levels resulted in a significant increase in the survival of these animals. Finally, we observed marked decreases in vascular endothelial growth factor, EphA2, and ephrin-B2 expression as well as marked decreases in angiogenesis in intestinal polyps in Apc(Min/+) c-myc(+/-) mice. This study shows that c-Myc is critical for Apc-dependent intestinal tumorigenesis in mice and provides a potential therapeutic target in the treatment of colorectal cancer.     

Effect of exercise on biological pathways in ApcMin/+ mouse intestinal polyps

Many epidemiological studies have demonstrated that level of exercise is associated with reduced colorectal cancer risk. Treadmill training can decrease Apc(Min/+) mouse intestinal polyp number and size, but the mechanisms remain unclear. Understanding the molecular changes in the tumor following exercise training may provide insight on the mechanism by which exercise decreases Apc(Min/+) mouse polyp formation and growth. The purpose of this study was to determine if exercise can modulate Apc(Min/+) mouse intestinal polyp cellular signaling related to tumor formation and growth. Male Apc(Min/+) mice were randomly assigned to control (n = 20) or exercise (n = 20) treatment groups. Exercised mice ran on a treadmill at a moderate intensity (18 m/min, 60 min, 6 days/wk, 5% grade) for 9 wk. Polyps from Apc(Min/+) mice were used to quantify markers of polyp inflammation, apoptosis, and beta-catenin signaling. Exercise decreased the number of macrophages in polyps by 35%. Related to apoptosis, exercise decreased the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells by 73% in all polyps. Bax protein expression in polyps was decreased 43% by exercise. beta-Catenin phosphorylation was elevated 3.3-fold in polyps from exercised mice. Moderate-intensity exercise training alters cellular pathways in Apc(Min/+) mouse polyps, and these changes may be related to the exercise-induced reduction in polyp formation and growth.     

Toll‐like receptor 4 regulates spontaneous intestinal tumorigenesis by up‐regulating IL‐6 and GM‐CSF

Inflammation is as an important component of intestinal tumorigenesis. The activation of Toll‐like receptor 4 (TLR4) signalling promotes inflammation in colitis of mice, but the role of TLR4 in intestinal tumorigenesis is not yet clear. About 80%–90% of colorectal tumours contain inactivating mutations in the adenomatous polyposis coli (Apc) tumour suppressor, and intestinal adenoma carcinogenesis in familial adenomatous polyposis (FAP) is also closely related to the germline mutations in Apc. The Apc^Min/+ (multiple intestinal neoplasia) model mouse is a well‐utilized model of FAP, an inherited form of intestinal cancer. In this study, Apc^Min/+ intestinal adenoma mice were generated on TLR4‐sufficient and TLR4‐deficient backgrounds to investigate the carcinogenic effect of TLR4 in mouse gut by comparing mice survival, peripheral blood cells, bone marrow haematopoietic precursor cells and numbers of polyps in the guts of Apc^Min/+ WT and Apc^Min/+ TLR4^?/? mice. The results revealed that TLR4 had a critical role in promoting spontaneous intestinal tumorigenesis. Significant differential genes were screened out by the high‐throughput RNA‐Seq method. After combining these results with KEGG enrichment data, it was determined that TLR4 might promote intestinal tumorigenesis by activating cytokine‐cytokine receptor interaction and pathways in cancer signalling pathways. After a series of validation experiments for the concerned genes, it was found that IL6, GM‐CSF (CSF2), IL11, CCL3, S100A8 and S100A9 were significantly decreased in gut tumours of Apc^Min/+ TLR4^?/? mice compared with Apc^Min/+ WT mice. In the functional study of core down‐regulation factors, it was found that IL6, GM‐CSF, IL11, CCL3 and S100A8/9 increased the viability of colon cancer cell lines and decreased the apoptosis rate of colon cancer cells with irradiation and chemical treatment.     

Myofiber degeneration/regeneration is induced in the cachectic ApcMin/+ mouse.

Cachexia is characterized as an inflammatory state induced by the cancer environment, which is accompanied by the loss of muscle and fat mass. Well-investigated mechanisms of cachexia include the suppression of myofiber protein synthesis and the induction of the protein degradation. However, it is not well characterized whether chronic inflammation during cachexia induces myofiber degeneration, which contributes to muscle mass loss and decreased functional capacity. The purpose of this study was to determine whether Apc(Min/+) mice, which demonstrate a chronic systemic inflammatory state due to an intestinal tumor burden, undergo cachexia and whether the myofibers exhibit signs of degeneration and/or regeneration. Six-month-old female Apc(Min/+) body weight decreased 21% compared with C57BL/6 mice and was not the result of blunted growth. Apc(Min/+) gastrocnemius muscle was reduced 45%, and soleus mean fiber cross-sectional area decreased 24% vs. C57BL/6 mice. Soleus muscle morphology demonstrated pathology of myofibers undergoing degeneration and/or regeneration. These data demonstrate that the Apc(Min/+) mouse becomes cachectic by 6 mo of age and that skeletal muscle degeneration and regeneration may be related to the muscle loss.     

Integrated analysis of the cerebrospinal fluid peptidome and proteome

Cerebrospinal fluid (CSF) is the only body fluid in direct contact with the brain and thus is a potential source of biomarkers. Furthermore, CSF serves as a medium of endocrine signaling and contains a multitude of regulatory peptides. A combined study of the peptidome and proteome of CSF or any other body fluid has not been reported previously. We report confident identification in CSF of 563 peptide products derived from 91 precursor proteins as well as a high confidence CSF proteome of 798 proteins. For the CSF peptidome, we use high accuracy mass spectrometry (MS) for MS and MS/MS modes, allowing unambiguous identification of neuropeptides. Combination of the peptidome and proteome data suggests that enzymatic processing of membrane proteins causes release of their extracellular parts into CSF. The CSF proteome has only partial overlap with the plasma proteome, thus it is produced locally rather than deriving from plasma. Our work offers insights into CSF composition and origin.     

Apc+/Min colonic epithelial cells express TNF receptors and ICAM-1 when they are co-cultured with large intestine intra-epithelial lymphocytes

Adenomatous polyposis coli (APC) functions are involved in the heterotypic interactions occurring between intestinal epithelial cells (IECs) and intra-epithelial lymphocytes (IELs). These interactions may be of interest in cancer prevention, since recent data provide evidence for lymphocyte mediated immunosurveillance of epithelial cancers. The present study attempts to determine if APC inactivation induces changes in the cross-talk between IEC and large intestine IEL (LI-IEL) through intercellular adhesion molecule (ICAM-1)/leukocyte function-associated (LFA-1) interactions. Mouse Apc+/+ and Apc+/Min colonocytes were co-cultivated with LI-IEL. When co-cultured with LI-IEL Apc+/Min IEC but not Apc+/+ IEC expressed high levels of ICAM-1. The presence of ICAM-1 was linked to TNFalpha production in both co-cultures and TNFR expression only in co-cultivated Apc+/Min IEC. Finally, butyrate enhanced the expression of ICAM-1 in Apc+/Min IEC co-cultured with LI-IEL, and the secretion of TNFalpha by both types of co-cultures. These events could participate in determining the Apc+/Min IEC immunogenicity under different in vivo conditions.     

Bortezomib and sphingosine kinase inhibitor interact synergistically to induces apoptosis in BCR/ABl+ cells sensitive and resistant to STI571 through down-regulation Mcl-1

IL-17 plays an important role in gut homeostasis. However, the role of IL-17F in intestinal tumorigenesis has not been addressed. Here we demonstrate that ablation of IL-17F significantly inhibits spontaneous intestinal tumorigenesis in the small intestine of Apc(Min/+) mice. IL-17F ablation decreased IL-1β and Cox-2 expression as well as IL-17 receptor C (IL-17RC) expression, which were increased in tumors from Apc(Min/+) mice. Lack of IL-17F did not reverse the splenomegaly but partially restored thymic atrophy, suggesting a local effect of IL-17F in the intestine. IL-17F deficient Apc(Min/+) mice showed a significant decrease in immune cell infiltration in the lamina propria. Interestingly, the expression of IL-17A from CD4 T cells in the lamina propria remains unchanged in the absence of IL-17F. Collectively, our results suggest the proinflammatory and essential role of IL-17F to develop spontaneous intestinal tumorigenesis in Apc(Min/+) mice in the presence of IL-17A.     

Quantitative analysis of the intra- and inter-individual variability of the normal urinary proteome

Urine is a readily and noninvasively obtainable body fluid. Mass spectrometry (MS)-based proteomics has shown that urine contains thousands of proteins. Urine is a potential source of biomarkers for diseases of proximal and distal tissues but it is thought to be more variable than the more commonly used plasma. By LC-MS/MS analysis on an LTQ-Orbitrap without prefractionation we characterized the urinary proteome of seven normal human donors over three consecutive days. Label-free quantification of triplicate single runs covered the urinary proteome to a depth of more than 600 proteins. The median coefficient of variation (cv) of technical replicates was 0.18. Interday variability was markedly higher with a cv of 0.48 and the overall variation of the urinary proteome between individuals was 0.66. Thus technical variability in our data was 7.5%, whereas intrapersonal variability contributed 45.5% and interpersonal variability contributed 47.1% to total variability. Determination of the normal fluctuation of individual urinary proteins should be useful in establishing significance thresholds in biomarker studies. Our data also allowed definition of a common and abundant set of 500 proteins that were readily detectable in all studied individuals. This core urinary proteome has a high proportion of secreted, membrane, and relatively high-molecular weight proteins.