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Preclinical data, and an increasing list of clinical investigations, show anti-inflammatory agents to favourably influence the biology of colorectal tumor. We have earlier reported on re-expression of activated immune cells after three days preoperative treatment of patients with colorectal carcinoma, randomized to receive oral NSAID (indomethacin or celebrex). Antisecretory prophylaxis (esomeprasol) was provided to all patients and served as sham treatment. Concomittant to MHC locus activation, Prominin1/CD133, a marker associated with stemness and poor prognosis in several solid tumors, was downregulated. The aim of the present study was to evaluate expression of additional regulators belonging to the stem cell niche, OCT4, SOX2 and BMP7, as well as some microRNAs, reported to act as tumor suppressors or oncomiRs. Peroperative tumor biopsies were analyzed by microarrays, quantitative real-time PCR and immunohistochemistry (IHC). The stem cell master regulator SOX2 was increased by NSAIDs (p

Background: Sporadic colorectal tumors probably carry genetic alterations that may be related to familiar clusters according to risk loci visualized by SNP arrays on normal tissues. The aim of the present study was therefore to search for DNA regions (copy number variations, CNVs) as biomarkers associated to genetic susceptibility for early risk predictions of colorectal cancer. Such sequence alterations could provide additional information on phenotypic grouping of patients. Material and Methods: High resolution 105K oligonucleotide microarrays were used in search for CNV loci in DNA from tumor-free colon mucosa at primary operations for colon cancer in 60 unselected patients in comparison to DNA in buffy coat cells from 44 confirmed tumor-free and healthy blood donors. Array-detected CNVs were confirmed by Multiplex ligation-dependent probe amplification (MLPA). Results: A total number of 205 potential CNVs were present in DNA from colon mucosa. 184 (90%) of the 205 potential CNVs had been identified earlier in mucosa DNA from healthy individuals as reported to the Database of Genomic Variants. Remaining 21 (10%) CNVs were potentially novel sites. Two CNVs (3q23 and 10q21.1) were significantly related to colon cancer, but not confirmed in buffy coat DNA from the cancer patients. Conclusion: Our study reveals two CNVs that indicate increased risk for colon cancer; these DNA alterations may have been acquired by colon stem cells with subsequent appearance among epithelial mucosa cells. Impact: Certain mucosa CNV alterations may indicate individual susceptibility for malignant transformation in relationship to intestinal toxins and bacterial growth.

Background : In asthma, clinical measures like FEV1 are a poor surrogate for detection of sub-groups and disease progression, and more efficient screening methods are needed. The PExA method (particles in exhaled air) non-invasively counts and collects exhaled particles, which reflect the composition of the fluid lining the peripheral airways and contains possible information about lung status. Collected particle mass is in the ng range and currently only single protein analysis is carried out routinely. The pilot study aimed to investigate Multiplex technology for simultaneous analysis of acute-phase proteins in exhaled particles. Method : Particles were collected during 30 minutes from two groups: Population 1: 12 healthy volunteers. Population 2: 6 subjects with asthma and 6 age-matched controls. Albumin, fibronectin, fibrinogen, SAP, A2M, CRP, haptoglobin and AGP were analysed using two Multiplex-kits (MILLIPLEX® SKINMAG-50K, MILLIPLEX® HCVD3-67K). Results: Albumin, fibrinogen, fibronectin, haptoglobin and AGP could be quantified in the majority of samples (>60%) and A2M in 7 samples, all regardless of subject9s health status. CRP and SAP were detected in one asthmatic and one control, respectively. Median particle mass collected in Population 1 was 340 ng (143-739). In Population 2, sampled mass in asthmatics (median 295 ng, 116-430) was significantly decreased compared to controls (median 535 ng, 234-1060) (P Conclusion : Multiplex technology can improve analysis possibilities by facilitating simultaneous quantification of multiple proteins in exhaled particles. Asthma may lead to a decrease in exhaled particles and less sample material.

Results from tests on the Multicentre Evaluation of In Vitro Cytotoxicity (MEIC) reference chemicals 31–50 in 67 different in vitro toxicity assays are presented in this paper as a prerequisite to in vitro/in vivo comparisons for all MEIC in vitro toxicity data in forthcoming papers, i.e. the final MEIC evaluation of the relevance of the tests. With the aim of increasing knowledge about the relative significance of some in vitro methodological factors, the strategies and methods of the preceding parts in the MEIC series (Parts II and III) were again employed to enable comparative cytotoxicity analysis of the new in vitro results presented in this paper. A principal components analysis (PCA) of the results from tests of the 20 chemicals in 67 assays demonstrated a dominating first component describing as much as 74% of the variance in the toxicity data, indicating a similar ranking of the cytotoxicities of the chemicals in most of the tests. The influence on the general variability of the results of a few, key methodological factors was also evaluated by using linear regression comparisons of the results of all pairs of methods available in the study, i.e. methods which were similar in all respects except for the factor being analysed. Results from this “random probe” analysis were: a) the cytotoxicities of 11 of the 20 chemicals increased considerably with exposure time (> 10 times over 4–168 hours); b) in general, human cell line toxicity was well predicted by cytotoxicity in animal cells; c) prediction of human cell line toxicity by most ecotoxicological tests was only fairly good; d) 14 comparisons of similar assays with different cell lines showed similar toxicities (mean R2 = 0.83); e) nine comparisons of similar assays employing different primary cultures and cell lines shared similar toxicities (mean R2 = 0.71); and f) 16 comparisons of similar assays with different growth/viability endpoints showed similar toxicities (mean R2 = 0.71). Results b, d, e and f must contribute to the PCA-documented high general similarity of the in vitro toxicity data. Results a and c, together with factors which were not analysed, such as different protocols and inter-laboratory variability of tests, could explain the 26% dissimilarity. To provide background information to the planned final MEIC evaluation of the relevance of the 61 methods in which all 50 chemicals have been tested, an additional PCA was made of the 50 chemical-61 assay in vitro database (from Parts II and III and the present paper). This supplementary PCA demonstrated an 80% similarity of results. Compared with the previous analysis of the tests of the first 30 MEIC reference chemicals (MEIC Part III), the present analysis of the tests of the last 20 MEIC chemicals indicates a somewhat higher variation in the results. Correspondingly, some deviating endpoint measurements and cell line responses were demonstrated by the pairwise comparisons in the present study. As a result, the analysis revealed a high correlation (R2 = 0.73) between the average human cell line toxicity and the results from a new protein denaturation test. These preliminary results suggest that intracellular protein denaturation may be a frequently occurring mechanism in basal cytotoxicity.

During 1988-1992, 684 scats were collected throughout the year in the territory of the only reproducing family group (mean five individuals) of wolves Canis lupus in Scandinavia. Moose Alces alces, roe deer Capreolus capreolus, and badger Meles meles constituted the three most important prey species, and hair from them was found in 52%, 50%, and 19% of scats, respectively. When compensating for different area/volume ratios in prey species of different size, these three species were estimated to constitute 97% of the biomass ingested. The proportions of moose, roe deer, and badger were 66%, 27%, and 8% by mass, and 25%, 52%, and 23% by number, respectively. Young-of-the-year dominated two samples of dead moose (51% of 65 killed by wolves; 43% of 155 killed by hunters), but no significant differences between the samples were found in any age class. Wolves killed significantly more female moose (76%) than hunters (53%), and among wolf-predated moose, no male was older than two years. Mean winter density of moose and roe deer in the wolf territory (523 km^2), estimated by fecal pellet group counts, was 1.5 moose and 0.4 roe deer/km^2. Moose density decreased slightly at the end of the study, but it was estimated that wolves killed only about 5% of the moose population each year and that this could be compensated for by a decrease of about 10-20% in the hunter kill. In spite of a high predation pressure from wolves, in addition to predation from an increasing lynx Lynx lynx population, the density of roe deer increased threefold. It is concluded that the future predation pressure on moose may be more pronounced if the density of wolves increases, and roe deer may be more affected by predation when the present favourable ecological conditions cease.

Expression of Prominin 1/CD133 is associated with poor prognosis and chemoresistance in several types of solid tumors. The aim of the present study was, therefore, to evaluate Prominin 1/CD133 expression in colorectal carcinoma after short-term preoperative treatment by non-steroidal anti-inflammatory drugs (NSAIDs). Patients aimed at curative operation for colon cancer were randomized to receive NSAIDs (indomethacin 50 mg x2 or celecoxib 100 mg x2) three days preoperatively. Antisecretory prophylaxis (esomeprasol 40 mg x1) was provided to all patients and served as sham intake. CD133 expression in tumor tissue was also assessed in tumors from Dukes' B patients selected for either long or short postoperative survival. No patients received perioperative chemoradiotherapy. Tumor tissue was collected at surgery for quantification of mRNAs (Prom1 and Wnt4) by qPCR. Immunohistochemistry stained for CD133, Ep-CAM, CD34 and CD45. PGE2 content in tumor tissue was determined. Transcript of CD133 in tumor tissue was lower in patients treated with NSAIDs (0.28 ± 0.07 vs. 0.51 ± 0.08; p

Non-steroidal anti-inflammatory drugs (NSAIDs) attenuate tumor net growth in clinical and experimental cancer. Evaluations in cell culture experiments have implied involvement of growth factor and G-protein related signaling pathways to explain decreased proliferation, angiogenesis, increased cell adhesion and apoptosis. Sparse information is however available from studies on growing tumors in vivo. The aim of the present study was to map alterations in selected signal proteins in relation to heterogeneous tissue expression of COX-2 in tumors during COX inhibition. MCG 101 cells were exposed to indomethacin treatment both in vivo and in vitro to reduce PGE 2 production. Tumor tissue specimens were taken for immunohistochemical analyses and qPCR determinations. Protein markers were selected to reflect cell proliferation and cell cycling, angiogenesis and metastasis in relationship to COX-2 staining in tumor tissue. Indomethacin did not change overall COX-2 staining in tumor tissue, but altered its distribution towards increased staining in cell nuclei/ nucleoli and decreased COX-2 staining heterogeneity in tumor tissue. P53 staining was decreased, while PCNA and TGFβ3 staining were increased by indomethacin in tumor areas with high presence of COX-2, which correlated to staining of BAX, TUNEL, Bcl-2, c-jun, p21, p27, p53 and NM23. Net tumor growth was predicted by EGF-R, p21 and p27 proteins in tumor tissue during indomethacin treatment (multivariate analysis). RNA transcript analyses showed decreased EGF-R and KRas expression in vivo, following indomethacin treatment, which also included KRas, PI3K, JAK1, STAT3 and c-jun, mRNAs in cultured tumor cells. In conclusion, our results extend earlier studies on cell culture experiments and demonstrate that EGF-R and downstream KRas pathways communicate effects of increased prostaglandin activity in tumor tissue in vivo.

This study shows that the Artemia assay, which is usually performed by incubation for a 24-hour period in artificial sea water, can also be performed in phosphate buffered saline (PBS) at pH 7.2, or in a cell culture medium, both of which are used in toxicity assays employing mammalian cells. Thus, by using the equivalent media for incubation, toxic effects in the Artemia assay and toxic mechanisms can more accurately be compared with results obtained in mammalian cell toxicity. Survival of control animals is good for 72 hours, provided that infection can be avoided. A pH greater than 6 is essential for good survival of Artemia Salina, and a pH greater than 10.5 should be avoided. Because of the risk of infection at low saline concentrations, a decreased incubation time of 16 or 12 hours is recommended. The lethal concentrations (LC10 and LC50) in the 24-hour Artemia assay of the first ten chemicals in the MEIC programme were measured in PBS, and the results compared with those from a previous study of the effects in a 10-minute acute ATP leakage assay, specifically indicating lesions in the cell membrane. The EC10 and EC50 values for the alcohols in the Artemia assay were 35–75% of the corresponding values in the ATP leakage assay. For paracetamol and amitriptyline, the EC10 and EC50 values in the Artemia assay were 2–16% of the corresponding values in the ATP-leakage assay. The greatly increased toxicity of the two compounds in the animals may be related to systemic effects. FeSO4 was very toxic to Artemia salina at a concentration of lug/ml, but it did not cause leakage of ATP from cultured cells, even at a concentration of 8,000μg/ml, showing that widely different mechanisms of interaction with FeSO4 are measured by the two assays. A lower toxicity of polygodial, a sesquiterpenoid unsaturated dialdehyde, in cell culture medium, was obvious in the Artemia assay. Thus, some factors in cell culture medium must, by interacting with the toxic molecule, protect the animals against the toxicity of polygodial, as was previously found in cultured cells.

1. 1. Diadenosine tetraphosphate (Ap4A) levels were determined in HL-60 cells differentiating into granulocytes or monocytes after treatment for 0–7 days with retinoic acid (RA) or 4-β-phorbol-12-myristate-13-acetate (PMA) respectively. 2. 2. The levels increased significantly compared to untreated control cells within 2 days and then declined again. 3. 3. In RA treated cells the levels finally decreased far below those of untreated HL-60 cells and became equal to those found in human granulocytes. 4. 4. PMA treatment had no effect on Ap4A levels in human granulocytes. 5. 5. A possible interaction between Ap4A and ADP-ribosyl transferase is discussed.

Previous studies in our laboratory have demonstrated that prostaglandin (PG) E2 is involved in anorexia/cachexia development in MCG 101 tumor-bearing mice. In the present study, we investigate the role of PGE receptor subtype EP2 in the development of anorexia after MCG 101 implantation in wild-type (EP2 (+/+)) or EP2-receptor knockout (EP2(-/-)) mice. Our results showed that host absence of EP2 receptors attenuated tumor growth and development of anorexia in tumor-bearing EP2 knockout mice compared to tumor-bearing wild-type animals. Microarray profiling of the hypothalamus revealed a relative twofold change in expression of around 35 genes including mRNA transcripts coding for Phospholipase A2 and Prostaglandin D2 synthase (Ptgds) in EP2 receptor knockout mice compared to wild-type mice. Prostaglandin D2 synthase levels were increased significantly in EP2 receptor knockouts, suggesting that improved food intake may depend on altered balance of prostaglandin production in hypothalamus since PGE2 and PGD2 display opposing effects in feeding control.

Ghrelin is a novel brain-gut peptide that stimulates food intake and may secondarily increase body weight via a growth hormone secretagogue receptor (GHS-R). Tumor-bearing mice (MCG101), characterized by anorexia, fat loss and muscle wasting due to increased concentration of PGE2 and proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha), were provided ghrelin i.p. at a low (20 microg/day) and high dose (40 microg/day) to examine the ability of ghrelin to counteract tumor-induced anorexia. Immunohistochemical staining and Western blot analyses were used to identify GHS-R expression in the brain as well as its relationship to NPY expression in hypothalamic neurons. GHS-R mRNA in hypothalamus and ghrelin mRNA in gastric fundus were quantified by RT-PCR. Body composition was determined by carcass extractions. GHS-R expression in hypothalamus and plasma ghrelin levels were significantly increased in freely-fed tumor-bearing mice, while gastric fundus expression of ghrelin was unaltered compared to non-tumor-bearing mice (controls). Ghrelin treatment increased food intake, body weight and whole body fat at both low and high doses of ghrelin in normal controls, while tumor-bearing mice showed improved intake and body composition at the high dose of ghrelin only. Exogenous ghrelin normalized the GHS-R expression in hypothalamus from tumor-bearing mice without alterations in the gastric fundus expression of ghrelin. Tumor growth was not altered by exogenous ghrelin. Our results indicate that MCG 101-bearing mice became ghrelin resistant despite upregulation of hypothalamic GHS-R expression, which confirms similar indirect observations in cancer patients. Thus, other factors downstream of the ghrelin-GHS-R system appear to be more important than ghrelin to explain cancer-induced anorexia.

Earlier observations on cyclo-oxygenase inhibitors (NSAIDs) restricting tumor growth were re-evaluated by comparing the effects of non-selective, preferential selective and selective derivatives of COX-inhibitors on tumor growth in mouse models with either prostaglandin-sensitive (MCG-101, human tumors) and -insensitive transplants (K1735-M2). Tumor growth, with and without provision of a classical cyclo-oxygenase inhibitor (indomethacin), was related to tumor content of COX-1/COX-2 protein as well as to EP1-EP4 and prostacyclin receptor expression. Mouse serum amyloid protein (SAP) was measured as an indicator of systemic inflammation, which relates to pro-inflammatory cytokines. Indomethacin inhibited tumor growth and prolonged the survival of mice bearing MCG-101 tumors, which display a high production of PGE2, while K1735-M2 tumors with insignificant amounts of PGE2 did not respond to indomethacin at all. However, the effects of various NSAIDs on tumor growth were highly variable in combination with the fact that most preferential selective and selective COX-2 inhibitors attenuated poorly systemic inflammation evaluated by plasma concentrations of mouse SAP. The ability of NSAIDs to attenuate tumor growth was not related to the tumor content of COX-2 protein as expected. Multivariate analysis suggests that significant COX-inhibition of tumor growth may be related to tumor expression of subtype EP2, EP3 (p

Preclinical and clinical studies in our laboratory have suggested that prostaglandin (PG) E2 is involved in anorexia and cachexia development, although the role of COX pathways on the pathogenesis of cancer cachexia remains to be clarified. Expressions of PGE (EP1, EP2, EP3alpha,beta,gamma and EP4) and PGI (IP) receptors in the central nervous system (brain cortex, hypothalamus and brain stem), in peripheral (liver, white adipose tissue and skeletal muscle) and tumor tissue from MCG-101-bearing mice with and without indomethacin treatment were investigated by RT-PCR and immunohistochemistry. Expression of EP1 in the liver and EP4 receptor in white adipose tissue were upregulated and responded to indomethacin treatment, while downregulated expression of EP3 in skeletal muscle from tumor-bearing mice was unresponsive to indomethacin treatment despite improved carcass weight. Expression of EP and IP receptors in brain and tumor tissue from tumor-bearing mice were neither related nor responsive to systemic PGE2 levels including increased IL-1beta, IL-6 and TNF-alpha host activities. The expression IP receptor in CNS, peripheral tissue and tumor tissue was unchanged by cachexia development. Our results suggest that transcription of EP receptors in liver, fat and skeletal muscle tissue may be a control level for host metabolic alterations during tumor progression, while overall EP and IP receptor expression in CNS did not indicate an important control level for appetite regulation in MCG 101-bearing mice despite prostanoid related anorexia.

We studied by microphysiometry functional effects of two different signalling molecules in the murine tumor cell lines, MCG 101 and K1735-M2, namely norepinephrine (NE) and prostaglandin E2 (PGE2). This methodology implies estimation of intracellular metabolism by measurements of extracellular acidification rate (ECAR). MCG 101 (an undifferentiated, epithelial-like tumor), in contrast to K1735-M2 (a melanoma), has been found to produce great amounts of PGE2. Challenge of MCG 101 cells with PGE2 (0.284 and 2.84 microM for 9 min) elicited an increase in ECAR by about 10 and 41% above basal level, respectively. Pretreatment with indomethacin (0.5 microM) reduced the response to the two PGE2 concentrations by about 70 and 25%, respectively. In contrast, PGE2 caused virtually no response in K1735-M2 cells. Moreover, NE caused increases in ECAR in both cell types, possibly via beta3-adrenoceptors, as investigated pharmacologically in MCG 101, and by immunocytochemistry in both cell lines. The results obtained strongly suggest functional receptors for PGE2 in MCG 101, but not K1735-M2 tumor cells. Functional receptors for NE were demonstrated in both cell lines. There is possibly an autocrine loop in the MCG 101 cells, in which PGE2 activates cyclooxygenase.

Mutations in p53 is a most common genetic alteration in human cancer and has been related to outcome, also in colorectal cancer. However, published results are not unanimous on this point without clear-cut explanations. Different analytical methods have been applied which could explain some discrepancies. Another factor of importance may be the source of nucleic acids used for identification of sequence alterations. Therefore we compared mutations in exons 7 and 8 of p53 found in genomic DNA, with mutations in cDNA from the same patients and evaluated to what extent LOH and mutations in p53 occurred simultaneously, which are prerequisites for a complete loss of p53 function according to the classic concept. cDNA and genomic DNA from tumor tissue from 123 patients were used. Thirty-four mutations were found in both tumor cDNA and genomic DNA. Twenty missense mutations were the same in both cDNA and genomic DNA. Two missense mutations, one 1 bp deletion and one nonsense mutation were only found in genomic DNA, while nine missense mutations and one 9 bp deletion were found in cDNA only. Statistical analysis showed no significant difference among mutations in cDNA and genomic DNA (p=1.00). Only 2 patients (5%) had both LOH and mutations in exons 2-11 of p53. One patient had LOH without p53 mutation, while 18 patients (44%) showed p53 mutation without LOH and 20 patients (49%) had wt p53 without LOH. Forty-four patients (52%) were non-informative for LOH of the entire cohort consisting of 85 patients. Our findings suggest that different results reported on the role mutated p53 may have in colorectal cancer are probably not explained by the source of nucleic acids (RNA vs DNA) for sequence determinations. Rather, unknown information about simultaneous but different presence of p53 LOH in previously published reports is more likely. However, an unexpectedly infrequent occurrence (5-10%) of complete and isolated p53 ablation (LOH + mutation) would demand considerably larger patient populations (1500-2000 patients) than ever published for relating p53 function to survival in patients with colorectal cancer. Therefore, the role of p53 dysfunction in progression of colorectal cancer remains uncertain.

It is well-recognized that cycloogygenase inhibitors attenuate tumor growth in tumor models, although underlying mechanisms are unclear. In the present study we report that indomethacin retards MCG-101 tumor growth on mice by induction of apoptosis/necrosis and inhibits telomere elongation. The inhibition of telomerase activity by NSAIDs (indomethacin, mobic, sulindac sulfone, suramin) was, however, not a universal finding, since a mouse melanoma (K1735-M2) did not respond. By contrast, a human cell line of colon carcinoma origin (HT-29), responded by both retarded growth and telomerase activity despite a low intrinsic production of prostaglandins, mainly PGE 2 . Therefore, it is not likely that indomethacin inhibition of tumor growth and telomere elongation is directly related to Cox-1/Cox-2 activities in tumor cells. Also, NSAIDs at 25 μM (sulindac sulfone) decreased growth and telomerase activity in MCG-101 cells, without any effects on PGE 2 production, while ibuprofen reduced PGE 2 production but had no effect on growth or telomerase activity. Our results demonstrate that cyclooxygenase inhibitors can retard tumor growth both in murine tumors and in human tumor cells by inhibition of telomerase activity in addition to previously recognized mechanisms as induction of apoptosis, inhibition of cell proliferation, influence on the expression of growth factors around growing tumors and attenuation of neoangiogenesis.

The role of amino acids, glucose and lipids in improving amino acid balance in peripheral tissues was evaluated. Primed constant infusion of L-[ring-2H5]phenylalanine in combination with flux measurements of glucose, free fatty acids (FFA) and amino acids across arm and leg tissues were applied in male volunteers after an overnight fast with subsequent primed constant infusions of amino acids (0.2 g N kg-1 body weight day-1), long-chain triglycerides (0.98-1.079 g kg-1 day-1) and glucose (3.13-3.62 g kg-1 day-1). Amino acids and phenylalanine tracer infusion continued for 6 h; the lipid infusion was provided during 2-6 h from the start, and glucose infusion was provided between 4 and 6 h. Flux measurements were performed at steady state before the next infusion started. Arterial concentrations of infused substrates increased during provision, but remained constant thereafter. Plasma insulin increased when glucose was provided, whereas insulin-like growth factor (IGF) I was unchanged during all infusions. Blood flow was unchanged in arm tissue during all infusions, while leg blood flow increased during fat and glucose infusion. FFA and glucose balance were unchanged during amino acid infusion but improved during lipid and glucose infusions. Amino acid balance was negative across arm and leg tissues in the fasted state, but reached balance during amino acid infusion. This effect was equally dependent on protein synthesis and protein degradation without any contribution from lipids and glucose. 3-Methylhistidine release from tissues was not influenced by any substrate. Our results suggest that extracellular amino acid concentrations determine amino acid balance across peripheral tissues independently of non-protein calories, insulin and IGF-I.

Rats were fed a 0% casein diet for 1 week, with or without enteral or parenteral administration of essential amino acids, or a 25% casein diet, in one group supplemented with 5-fluorouracil treatment. Ninety minutes before sacrifice the rats were given a tracer of [3H]orotic acid. Incorporation into the acid soluble fraction, RNA, and DNA was determined in liver, small intestine, bone marrow, and kidney. Nucleotide profile was examined in liver and intestine. Protein deficiency caused inter alia a decrease in body weight; a decrease in RNA/DNA ratio and an increase in the specific RNA labeling in liver and kidney; an altered nucleotide profile in the liver; an increase in the nucleotide/DNA and RNA/DNA ratios and a decrease in the specific labeling of the acid soluble fraction, RNA, and DNA in the bone marrow. These changes were prevented to the same extent by giving essential amino acids, either orally or intravenously. The minor changes in intestinal nucleotide profile in protein deprivation were prevented to a slightly larger extent by amino acids orally than parenterally. 5-Fluorouracil treatment gave a decrease in the RNA/DNA ratio in the liver and kidney but an increase in the nucleotide/DNA and RNA/DNA ratios in the bone marrow. Nucleotide profiles were unaltered. The amount of DNA per gram of tissue decreased in bone marrow and increased in kidney. Parenteral administration per se resulted in almost no changes.

Diadenosine tetraphosphate (Ap4A), a candidate for a signal molecule in the induction of DNA synthesis, was measured in regenerating livers of young adult rats at 12 and 24 h and of older rats at 24 h after partial hepatectomy. dATP and dTTP levels, which indicate the degree of proliferation in the livers, were determined by high-performance liquid chromatography and an enzymatic method, respectively. The Ap4A levels were increased in the beginning of DNA synthesis. In young rats the levels were about 140% of those of unoperated rats and in older rats about 300%. This increase was considerably smaller than that found in another study comprising two regenerating rat livers excised 20 h after partial hepatectomy, but still supports the hypothesis that Ap4A might take part in the onset of proliferation. The greater Ap4A increase in older rats may suggest a possible need for a stronger triggering mechanism to start proliferation in aged tissue. However, the experiments do not prove a function for Ap4A in the induction of DNA synthesis and it cannot be excluded that Ap4A is a product of an independent reaction.

1. The intracellular compartmentation of diadenosine tetraphosphate (Ap4A) and of dTTP was studied in rat liver cells using non-aqueous glycerol for the isolation of cell nuclei. 2. This method allows a stepwise removal of cytoplasm from the nuclei. 3. The decrease in Ap4A or dTTP during the process was compared to the simultaneous decrease in RNA, which was taken to represent the cytoplasm. 4. In regenerating liver excised 24 hr after partial hepatectomy, Ap4A was almost equally distributed between the nucleus and cytoplasm. 5. In livers from unoperated control rats, the nuclear concentration of Ap4A was slightly elevated compared to that of whole cells. dTTP was only investigated in regenerating liver. 6. Significantly higher concentrations were found in the nuclear fractions. 7. The purest nuclei contained about 26% of whole cell levels of dTTP, while their RNA values had decreased to 7% of the whole cell RNA. 8. Considering that the liver cell nucleus comprises about 7% of the entire cell mass, a nuclear dTTP concentration of 26% indicates significantly higher dTTP levels in the nuclear compartment than in the cytoplasm of regenerating rat liver cells.

Rat liver whole cells and cell nuclei were prepared by a non-aqueous technique (glycerol). The nuclear preparations were of different purity as determined by RNA/DNA ratios (0.17-1.60) and accordingly were divided into 3 subgroups (mean values 0.29, 1.04 and 1.48). RNA nucleotides were separated by isotachophoresis and HPLC and calculated per mg DNA. Two of the nuclear subgroups (RNA/DNA = 1.04 and 1.48) had significantly elevated nucleotide values in relation to RNA/DNA. UDP-N-acetylhexosamine/DNA, on the contrary, was reduced in conformity with RNA in the preparations. Our findings may indicate different nucleotide concentrations in different parts of the cell.

Background Increased cyclooxygenase activity promotes progression of colorectal cancer, but the mechanisms behind COX-2 induction remain elusive. This study was therefore aimed to define external cell signaling and transcription factors relating to high COX-2 expression in colon cancer tissue. Method Tumor and normal colon tissue were collected at primary curative operation in 48 unselected patients. COX-2 expression in tumor and normal colon tissue was quantified including microarray analyses on tumor mRNA accounting for high and low tumor COX-2 expression. Cross hybridization was performed between tumor and normal colon tissue. Methylation status of up-stream COX-2 promoter region was evaluated. Results Tumors with high COX-2 expression displayed large differences in gene expression compared to normal colon. Numerous genes with altered expression appeared in tumors of high COX-2 expression compared to tumors of low COX-2. COX-2 expression in normal colon was increased in patients with tumors of high COX-2 compared to normal colon from patients with tumors of low COX-2. IL1β, IL6 and iNOS transcripts were up-regulated among external cell signaling factors; nine transcription factors (ATF3, C/EBP, c-Fos, Fos-B, JDP2, JunB, c-Maf, NF-κB, TCF4) showed increased expression and 5 (AP-2, CBP, Elk-1, p53, PEA3) were decreased in tumors with high COX-2. The promoter region of COX-2 gene did not show consistent methylation in tumor or normal colon tissue. Conclusions Transcription and external cell signaling factors are altered as covariates to COX-2 expression in colon cancer tissue, but DNA methylation of the COX-2 promoter region was not a significant factor behind COX-2 expression in tumor and normal colon tissue.

Synthesis and degradation of globular and myofibrillar proteins across arm and leg muscles were examined during stepwise increased intravenous infusion of amino acids (0.1, 0.2, 0.4, and 0.8 g N.kg-1.day-1) to healthy volunteers. Protein dynamics were measured by a primed constant infusion of L-[ring-2H5]phenylalanine and the release of 3-methylhistidine from skeletal muscles. Arterial concentrations and flux of glucose, lactate, and free fatty acids were unchanged despite increasing concentrations of plasma amino acids from 2.6 to 5.7 mM. Plasma insulin, insulin-like growth factor I (IGF-I), and plasma concentrations of IGF-I-binding proteins-1 and -3 remained at fasting levels throughout the investigation. Amino acid infusion caused a significant uptake of the majority of amino acids across arm and leg tissues, except tyrosine, tryptophan, and cysteine, probably due to low concentrations of these amino acids in the formulation. The balance of globular proteins improved significantly (P < 0.01) due to stimulation of synthesis and attenuation of degradation across arm and leg tissues, despite insignificant uptake of tyrosine, tryptophan, and cysteine. Degradation of myofibrillar proteins was uninfluenced by provision of amino acids. The results demonstrate that neither insulin nor circulating IGF-I explained improved protein balance in skeletal muscles after elevation of plasma amino acids. Rather, some amino acids in themselves trigger cellular reactions that initiate peptide formation. Limited availability of some extracellular amino acids was overcome by increased reutilization of the intracellular amino acid.

Tumor-bearing mice with two different locally growing malignant tumors (epithelial like, MCG 101; malignant melanoma, K1735-M2) were used to evaluate the putative role of prostaglandins for survival and local tumor growth in experimental cancer. Daily systemic injections of indomethacin (1 mu g/g bw) were used to block prostaglandin production in normal and T-cell deficient tumor-bearing nude mice. Tumor progression was determined by measurements of tumor weight, DNA-synthesis, cell cycle kinetics in vivo and in vitro (flow cytometry), tumor tissue concentrations of polyamines (putrescine, spermidine, spermine) and tumor tissue gene expression of growth regulating factors (IL-1 alpha, IL-6, TNF alpha, A,B-PDGF, EGF, VEGF, bFGF, TGF beta(3), angiogenin and transferrin receptor). Tumor tissue content of von Willebrandt factor VIII was estimated by immunohistochemistry. Indomethacin had no effect on survival, host nutritional state or local tumor growth in mice bearing the malignant melanoma with low PGE(2) production. In contrast, indomethacin prolonged survival, improved cachexia and decreased tumor growth in mice bearing the MCG 101 tumor with hundredfold higher prostaglandin tumor production, leading to elevated liver and muscle tissue as well as plasma concentrations of PGE(2). Indomethacin inhibited almost completely the high tumor PGE(2) production in MCG tumors, leading to prolonged potential doubling time for tumor growth in vivo, and a trend to decreased tumor tissue concentration of polyamines (spermidine). Indomethacin had no inhibitory effect on tumor cell proliferation in vitro, although PGE(2) production was decreased by 75%. The effect of indomethacin in vivo was independent of T-cells and was observed with similar magnitude irrespective of the number of MCG cells (10(4)-10(6)) implanted or the site of implantation (s.c., i.p., liver, lung, skeletal muscles). Tumor growth inhibition by indomethacin was not intrinsically transferable by tumor cells from indomethacin treated tumor-animals. Tumor expression of mRNA for several growth regulating factors were either increased (IL-6, TNF alpha, GM-CSF, TGF beta(3)) unchanged (EGF, VEGF, PDGF A,B, IL-1 alpha, transferrin receptor) or decreased (b-FGF and angiogenin) (p