Genes with Relevance for Early to Late Progression of Colon Carcinoma Based on Combined Genomic and Transcriptomic Information from the Same Patients

Background: Genetic and epigenetic alterations in colorectal cancer are numerous. However, it is difficult to judge whether such changes are primary or secondary to the appearance and progression of tumors. Therefore, the aim of the present study was to identify altered DNA regions with significant covariation to transcription alterations along colon cancer progression. Methods: Tumor and normal colon tissue were obtained at primary operations from 24 patients selected by chance. DNA, RNA and microRNAs were extracted from the same biopsy material in all individuals and analyzed by oligo-nucleotide array-based comparative genomic hybridization (CGH), mRNA- and microRNA oligo-arrays. Statistical analyses were performed to assess statistical interactions (correlations, co-variations) between DNA copy number changes and significant alterations in gene and microRNA expression using appropriate parametric and non-parametric statistics. Results: Main DNA alterations were located on chromosome 7, 8, 13 and 20. Tumor DNA copy number gain increased with tumor progression, significantly related to increased gene expression. Copy number loss was not observed in Dukes A tumors. There was no significant relationship between expressed genes and tumor progression across Dukes A–D tumors; and no relationship between tumor stage and the number of microRNAs with significantly altered expression. Interaction analyses identified overall 41 genes, which discriminated early Dukes A plus B tumors from late Dukes C plus D tumor; 28 of these genes remained with correlations between genomic and transcriptomic alterations in Dukes C plus D tumors and 17 in Dukes D. One microRNA (microR-663) showed interactions with DNA alterations in all Dukes A-D tumors. Conclusions: Our modeling confirms that colon cancer progression is related to genomic instability and altered gene expression. How- ever, early invasive tumor growth seemed rather related to transcriptomic alterations, where changes in microRNA may be an early phenomenon, and less to DNA copy number changes

Initiation of Protein Biosynthesis in Skeletal Muscles at Feeding

Background and aim. Artificial nutrition by intravenous feeding has for decades indicated less than optimal support of whole-body protein metabolism and balance during some treatment conditions. Therefore, the present project was aimed to evaluate the role and effects by amino acid provision to skeletal muscle cells in the light of other known important factors as amino acid infusion kinetics, IGF-I and insulin in support of myofibrillar protein synthesis. Methods. Murine L6 and human rhabdomyosarcoma cells were cultured at standardized conditions in the presence of various amino acid concentrations. Commercially available amino acid formulations were infused by constant rates to patients scheduled for elective surgery and to ICU patients. Transgenic female mice with selective knockout of the IGF-I gene in hepatocytes were used in refeeding experiments to evaluate the role of circulating IGF-I for muscle protein synthesis, which was estimated by the flooding dose technique ([14C]-phenylalanine). Protein factors for translational control of protein synthesis and cell signaling (4E-BPI, eIF4E, p70s6k, mTOR) were estimated in Western blots. Transcripts of muscle IGF-I, IGF-IR, PI3-kinase, AKT, mTOR, acta 1 (alpha-actin), mhc2A (myosin) and slc38a2/Snat 2 (amino acid transporter) were quantified by qPCR. Plasma amino acids were measured by HPLC. Results. Incorporation rate of amino acids into muscle proteins gave incorrect results in a variety of experimental conditions. Methods independent of labeled amino acids (ribosome profiles, initiation factor analyses) indicated that essential amino acids activate initiation of protein translation, while non-essential amino acids had no such effects. Insulin at physiologic concentration (100 µM/ml) did not stimulate global muscle protein synthesis, but did so at supraphysiologic concentrations (3mU/ml). Circulating IGF-I was not critical for activation of muscle protein translation, while tissue produced IGF-I and IGF-IR controlled feeding induced protein synthesis, which involved mTOR signaling in skeletal muscles. In general, provision of exogenous amino acids was related to plasma concentrations and probably to steady state levels of amino acids in peripheral tissues of patients. Amino acids caused activation of translation initiation of muscle proteins as demonstrated for myosin heavy chain and -actin. These effects by amino acids were in part supported by increased transcription and utilization of amino acid transporters as Snat 2 mRNA in muscles. Microarray analysis indicated up-regulation of genes in the mevalonate-pathway following amino acid exposure, important for steroidogenesis and lipid metabolism, which may imply new and additional mechanisms behind anabolic reactions in muscle cells related to nutrition. Conclusion. Our results re-emphasize that labeled amino acids should be used with great caution for quantification of muscle protein synthesis. Measurements of regulating factors in the control of initiation of muscle protein synthesis represent alternative and convenient applications in estimation of directional changes in protein synthesis during non steady state conditions as demonstrated by overnight preoperative provision of standard TPN to patients. Our results confirm that muscle cells are sensitive to alterations in extracellular concentrations of amino acids, which signal to activate translation of transcripts for myofibrillar proteins. Such dynamics are highly dependent on the presence of membrane transporters of amino acids

Initiation of muscle protein synthesis was unrelated to simultaneously upregulated local production of IGF-1 by amino acids in non-proliferating L6 muscle cells.

BackgroundIGF-1 is considered an important regulator of muscle protein synthesis. However, its role in stimulation of muscle protein synthesis by amino acids (AA) is not clear, despite pronounced alterations in IGF-1 mRNA expression and signaling in muscle tissues by feeding. This study evaluates the role of locally produced IGF-1 and IGF-1 signaling when skeletal muscle protein synthesis is activated by increased amino acid availability in confluent, non-proliferating cells.MethodsL6 skeletal muscle cells were subjected to amino acid starvation (24 h, 0.14 mM) followed by 18 h amino acid refeeding in Low AA (0.28 mM) or High AA concentrations (9 mM). Protein synthesis rates were estimated by L-[U-14C]-phenylalanine incorporation into cellular proteins. IGF-1 and IGF-1 receptor mRNA expression were quantified by real time PCR. SiRNA knockdown, antibodies and chemical inhibitors were used to attenuate muscle IGF-1 production and signaling.ResultsHigh AA concentrations (9mM) increased IGF-1 mRNA expression (+ 30%, pConclusionAmino acids increased IGF-1 mRNA expression and stimulated muscle protein synthesis. However, simultaneous upregulation of IGF-1 mRNA did not relate to increased protein synthesis by amino acids. The results indicate that increased IGF-1 mRNA expression is rather a covariate to amino acid initiation of protein synthesis in non-proliferating muscle cells; effects that may be related to unrecognized metabolic activities, such as transport of amino acids

Pituitary adenylate cyclase-activating polypeptide 6-38 blocks cocaine- and amphetamine-regulated transcript Peptide-induced hypophagia in rats.

Cocaine- and amphetamine-regulated transcript peptides (CARTp) suppress nutritional intake after administration into the fourth intracerebral ventricle. Recent in vitro studies have shown that PACAP 6-38, a pituitary adenylate cyclase-activating polypeptide (PACAP) fragment, could act as a competitive antagonist against CARTp 55-102 on a common CARTp-sensitive receptor structure. Here, we show for the first time in vivo that the reduction in solid food intake induced by exogenous CARTp 55-102 (0.3 nmol: 1.5 µg) administered fourth i.c.v. is blocked by pretreatment with PACAP 6-38 (3 nmol). The PACAP 6-38 fragment had no effect by itself either when given into the fourth ventricle or subcutaneously. Although effective to block the CARTp-effect on feeding and short-term body weight, PACAP 6-38 failed to attenuate CARTp-associated gross motor behavioral changes suggesting at least two CARTp-sensitive receptor subtypes. In conclusion, PACAP 6-38 acts as a functional CARTp antagonist in vivo and blocks its effects on feeding and short term weight gain

Peripheral administration of PACAP 6-38 does not affect solid food intake.

<p>PACAP 6-38 (0.3–6 nmol) did not affect solid food intake after s.c. administration as compared to vehicle in Experiment 3. Cumulative solid food intake in rats (n = 7) expressed as mean (g) ±SE.</p

Initiation factors for translation of proteins in the rectus abdominis muscle from patients on overnight standard parenteral nutrition before surgery

A B S T R A C T Previous studies have provided conflicting conclusions concerning the efficacy of improving protein balance in patients by standard intravenous nutrition [TPN (total parenteral nutrition)], which is either explained by suboptimal nutritional regimens or insensitive clinical methods. The aim of the present study was therefore to evaluate the effects on the initiation of translation of skeletal muscle proteins by standard overnight TPN. A total of 12 patients who underwent standard surgery were included. TPN was provided as an all-in-one treatment by constant infusion [0.16 gN · kg −1 of body weight · day −1 (30 kcal · kg −1 of body weight · day −1 )]. Saline-infused patients served as controls. Rectus abdominis muscle biopsies were taken at the time of the operation. The phosphorylation state of the proteins for initiation of translation was quantified. Plasma glucose, and serum insulin, glycerol, triacylglycerols (triglycerides) and NEFAs (non-esterified fatty acids; &apos;free fatty acids&apos;) were not significantly altered during TPN infusion, whereas total plasma amino acids increased, as shown by increases in methionine, phenylalanine, threonine, alanine, arginine, aspartic acid, glycine and histidine (P &lt; 0.05). Overnight TPN increased the formation of active eIF4G-eIF4E (where eIF is eukaryotic-initiation factor) complexes (P &lt; 0.05), whereas the inhibitory complex 4E-BP1 (eIF4E-binding protein)-eIF4E was moderately decreased (P &lt; 0.06). TPN increased the amount of the most phosphorylated form of 4E-BP1 (P &lt; 0.05), and increased the amount (P &lt; 0.04) and phosphorylation (P &lt; 0.01) of p70 S6K (70 kDa ribosomal protein S6 kinase). In conclusion, an overnight pre-operative constant infusion of standard TPN altered initiation factor complexes, indicating activation of the initiation of protein translation in rectus abdominis muscle in the presence of increased plasma amino acid levels, but without a concomitant increase in energy substrates and insulin. In contrast with our results from previous studies, the methodology used in the present study appears to be more sensitive in reflecting directional changes in human muscle protein synthesis compared with traditional methods, particularly based on measurements of amino acid flux

Fourth i.c.v. administration of PACAP 6-38 does not by itself affect solid food intake.

<p>PACAP 6-38 (0.3–3 nmol) did not affect food intake by itself vs. vehicle after fourth i.c.v. administration in Experiment 2. Cumulative solid food intake in rats (n = 8) expressed as mean (g ±SE).</p

Fourth i.c.v. PACAP 6-38 blocks CARTp-induced reductions in solid food intake.

<p>Male Sprague-Dawley rats (n = 8) were pretreated with varying doses of PACAP fragment 6-38 (range 0.3–3 nmol) or vehicle prior to dosing with 0.3 nmol CARTp or vehicle. (A) Solid food intake measures (mean g ±SE) from the entire 22 h observation period. Cumulative solid food intake was recorded at 2 h (B), 5 h (C), and 22 h (D) following peptide injections and food presentation. CARTp significantly inhibited food intake 2–22 h after injection (Fig. 1A–D). Pretreatment with 3 nmol PACAP 6-38 completely blocked the effect after 5 h (Fig. 1 C). Significances from Tukey’s post hoc test after repeated measures ANOVA are indicated above: *p<0.05; **p<0.01; ***p<0.001. The treatment abbreviations are V = saline vehicle; C = 0.3 nmol CARTp; P_0.3 = 0.3 nmol PACAP 6-38; P_0.6 = 0.6 nmol PACAP 6-38; P₃ = 3 nmol PACAP 6-38.</p

PACAP 6-38 blocks CARTp-induced reduction in body weight.

<p>Effects by fourth i.c.v. drug treatment on body weight changes (mean g ± SE). (A) The animals receiving CARTp had significantly decreased body weight on the day after fourth i.c.v. injections (22 h post injection); however, PACAP 6-38 (P) was able to mitigate the weight loss effects observed in the animals. (B) During the 24 h preceding the next treatment session (i.e., 2 days later), all animals again displayed stable positive weight changes that were similar across groups, regardless of the preceding treatments. Significance from Tukey’s post-tests after repeated measures ANOVA are indicated: *p<0.05; **p<0.01; ***p<0.001. All significant differences shown in the figure are vs. the vehicle control condition.</p

Host knockout of E-prostanoid 2 receptors reduces tumor growth and causes major alterations of gene expression in prostaglandin E2-producing tumors

Prostaglandin E-2 (PGE(2)) is elevated in a variety of malignant tumors and has been shown to affect several hallmarks of cancer. Accordingly, the PGE, receptor, E-prostanoid 2 (EP2), has been reported to be associated with patient survival and reduced tumor growth in EP2-knockout mice. Thus, the aim of the present study was to screen for major gene expression alterations in tumor tissue growing in EP2-knockout mice. EP2-knockout mice were bred and implanted with EP2 receptor-expressing and PGE(2)-producing epithelial-like tumors. Tumor tissue and plasma were collected and used for analyses with gene expression microarrays and multiplex enzyme-linked immunosorbent assays. Tumor growth, acute phase reactions/systemic inflammation and the expression of interleukin-6 were reduced in EP2-knockout tumor-bearing mice. Several hundreds of genes displayed major changes of expression in the tumor tissue when grown in EP2-knockout mice. Such gene alterations involved several different cellular functions, including sternness, migration and cell signaling. Besides gene expression, several long non-coding RNAs were downregulated in the tumors from the EP2-knockout mice. Overall, PGE(2) signaling via host EP2 receptors affected a large number of different genes involved in tumor progression based on signaling between host stroma and tumor cells, which caused reduced tumor growth.Funding Agencies|Swedish Cancer Society [CAN 2010/255]; Swedish State under the LUA/ALF agreement; Assar Gabrielsson foundation; Magnus Bergvall foundation</p