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Background: Red meat intake has been linked to increased colorectal cancer risk but the underlying mechanisms remain unclear. Experimental studies suggest a role for dietary heme, but epidemiological evidence for its carcinogenic potential is inconclusive. Because heme may promote specific mutations, it could be insightful to link heme exposure data to colorectal cancer (CRC) with such mutations in key genes in an observational, population-based study. Methods: We investigated the association between dietary heme iron intake (tertiles) and CRC cancer with adenomatous polyposis coli (APC) and Kirsten ras (KRAS) mutations, and tumor protein 53 (TP53) overexpression, in the Netherlands Cohort Study. After 7.3 yrs of follow-up, excluding the first 2.3 years, adjusted rate ratios (RR) and 95% confidence intervals (CI) were calculated, using 3,949 subcohort members (aged 55-69 years at baseline) and 733 CRC patients. Results: Heme iron intake was associated with an increased risk of CRC with activating G>A mutations in KRAS (RR=2.14 for the highest compared to the lowest intake; 95% CI: 1.17, 3.92; Ptrend = 0.01). In addition, a positive association between heme intake and the risk of overall G>A mutations in APC tumors was observed (RR=1.73 for the highest compared to the lowest intake; 95% CI: 1.18, 2.54; Ptrend = 0.005). However, this association was not found when restricting our analyses to CRC harboring G>A mutations leading to a truncated APC (RR=1.35 for the highest compared to the lowest intake; 95% CI: 0.79,2.37; Ptrend = 0.29). Moreover, there was a positive association between heme iron intake and the risk of P53 overexpressed tumors which was not observed in tumours without P53 overexpression (Pheterogeneity = 0.08). Conclusion: These results suggest that heme iron intake is associated with an increased risk of colorectal tumors harboring G>A transitions in KRAS and to a lesser extend in APC, and overexpression of P53. Taking CRC heterogeneity into account contributes to a better molecular understanding of the established association between red meat intake and CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 637. doi:1538-7445.AM2012-637

Thrombosis of the umbilical cord vessels is a rare but life-threatening event, usually leading to the death of the fetus. This report presents a case of an intra-partum fetal death due to thrombosis of the ductus venosus, hitherto not described in the literature. Labour was induced with intravenous oxytocin in a 21-year-old nulliparous woman because of postmaturity at 43 weeks gestation. Abrupt fetal bradycardia developed at 5 cm dilatation and fetal demise occurred within minutes without any sign of abruptio placentae or uterine hyperactivity. A cesarean section was performed because of failure to deliver the macrosomic fetus by vacuum extraction. On autopsy a fresh occlusive thrombus was found at the beginning of the ductus venous in the portal sinus. Maternal and fetal risk factors known to be associated with umbilical cord thrombus formation were excluded. Repeated assessment of Bishop scores is thought to have caused local infection of the membranes at the internal cervical os, resulting in general amnionitis and vasculitis of the umbilical cord. Secondary to the infection thrombus formation took place either primarily in the portal sinus and ductus venosus or in the umbilical vein with subsequent detachment and embolisation into the portal sinus, therewith occluding the ductus venosus leading to fetal death.

Endocrine cells occur in approximately 30% of all colorectal adenocarcinomas, and this feature appears to correlate with a relatively poor prognosis. To study the factors regulating endocrine differentiation in colorectal cancer, which may bear resemblance to the regulation of endocrine differentiation in normal intestinal mucosa, models in which differentiation can be manipulated are essential. However, endocrine features in colorectal cancer cell lines are scarce and are almost exclusively observed in xenografts, presumably as a result of differentiation induction by stromal components. We attempted to demonstrate endocrine differentiation in the colonic adenocarcinoma cell line Caco-2, which is frequently used as a model for enterocytic differentiation. In vitro endocrine tumor cells were not encountered. In vivo studies were cumbersome, because of the low take rate of Caco-2 cells. We did manage to establish nude mouse xenografts of Caco-2 cells by inoculating cells in collagen gel and by suppressing natural killer cell activity. In an attempt to induce a better take rate and to investigate the effect of Ras oncoprotein overexpression on endocrine differentiation, Caco-2 cells were transfected with a point-mutated c-Ha-Ras gene. The cell line Caco-2 EJ6, generated from these experiments, could be xenografted in nude mice with a high take rate, yielding a moderately well differentiated adenocarcinoma, morphologically identical to the tumors derived from untransfected Caco-2 cells. The xenografts displayed goblet cell, enterocytic, Paneth cell and endocrine differentiation. In vitro endocrine differentiation was observed neither under standard conditions nor with extracellular matrix components as differentiation inducers. We conclude that the Caco-2 cell line and its c-Ha-Ras transfected subline Caco-2 EJ6 in vivo display endocrine differentiation. Ras overexpression does not enhance endocrine differentiation. Due to its favorable growth properties in vivo, Caco-2 EJ6 is a suitable model for studies on endocrine differentiation in colorectal cancer.

Background & Aims Histone deacetylases (HDACs) are important regulators of chromatin involved in silencing tumor suppressor genes. We examined mutation of HDAC2 and examined consequences of HDAC2 loss. Methods Colon cancer cell lines and primary cancers were examined for mutations in HDAC2 by direct sequencing and capillary electrophoresis. Promoter methylation was determined using methylation-specific polymerase chain reaction in primary tumors. Sensitivity to HDAC inhibitors and resistance in vitro used colon cancer cell lines. Results HDAC2 mutations in the poly(A) tract in exon 1 result in a frameshift and premature stop codon. These were found in microsatellite instability (MSI) cell lines and 43% of MSI colon cancers, but only 7% of microsatellite stable cancers. Mutation was associated with complete or regional tumor specific loss of HDAC2 protein. Inactivation of HDAC2 was not associated with large-scale changes in promoter region methylation, but rather is a consequence of epigenetic MLH1 inactivation leading to MSI. HDAC2 mutation results in apoptotic resistance to HDAC inhibitors (trichostatin A or suberoylanilide hydroxamic acid), despite induction of global histone acetylation. Differential induction of apoptosis by HDAC inhibitors is mediated by the proapoptotic gene APAF1 , as shown by specific APAF1 induction only in cell lines with functional HDAC2, HDAC2 protein localization to the APAF1 promoter by chromatin immunoprecipitation, siRNA knockdown of HDAC2 leading to up-regulation of APAF1 , and stable knockdown of APAF1 reducing apoptotic response to HDAC inhibitors. Conclusions Frequent HDAC2 mutations are found in MSI tumors and HDAC2 plays a major role in mediating apoptotic response to HDAC inhibitors through direct regulation of APAF1.

BACKGROUND: Intestinal ischemia-reperfusion (IR) is a phenomenon related to physiological conditions (e.g. exercise, stress) and to pathophysiological events (e.g. acute mesenteric ischemia, aortic surgery). Although intestinal IR has been studied extensively in animals, results remain inconclusive and data on human intestinal IR are scarce. Therefore, an experimental harmless model for human intestinal IR was developed, enabling us to clarify the sequelae of human intestinal IR for the first time. METHODS AND FINDINGS: In 30 patients undergoing pancreatico-duodenectomy we took advantage of the fact that in this procedure a variable length of jejunum is removed. Isolated jejunum (5 cm) was subjected to 30 minutes ischemia followed by reperfusion. Intestinal Fatty Acid Binding Protein (I-FABP) arteriovenous concentration differences across the bowel segment were measured before and after ischemia to assess epithelial cell damage. Tissue sections were collected after ischemia and at 25, 60 and 120 minutes reperfusion and stained with H&E, and for I-FABP and the apoptosis marker M30. Bonferroni's test was used to compare I-FABP differences. Mean (SEM) arteriovenous concentration gradients of I-FABP across the jejunum revealed rapidly developing epithelial cell damage. I-FABP release significantly increased from 290 (46) pg/ml before ischemia towards 3,997 (554) pg/ml immediately after ischemia (p

Background The early to intermediate stages of the majority of colorectal tumours are thought to be driven by aberrations in the Wnt (APC, CTNNB1) and Ras (K-ras) pathways. A smaller proportion of cancers shows mismatch repair deficiency. The aim of this study was to analyse the co-occurrence of these genetic alterations in relation to tumour and patient characteristics. Methods In a group of 656 unselected sporadic colorectal cancer patients, aberrations in the APC, K-ras, CTNNB1 genes, and expression of hMLH1 were investigated. Additionally, tumours were divided in groups based on molecular features and compared with respect to patient's age at diagnosis, sex, family history of colorectal cancer, tumour sub-localisation, Dukes' stage and differentiation. Results Mutations at the phosphorylation sites (codons 31, 33, 37, and 45) in the CTNNB1 gene were observed in tumours from only 5/464 patients. Tumours with truncating APC mutations and activating K-ras mutations in codons 12 and 13 occurred at similar frequencies (37% (245/656) and 36% (235/656), respectively). Seventeen percent of tumours harboured both an APC and a K-ras mutation (109/656). Nine percent of all tumours (58/656) lacked hMLH1 expression. Patients harbouring a tumour with absent hMLH1 expression were older, more often women, more often had proximal colon tumours that showed poorer differentiation when compared to patients harbouring tumours with an APC and/or K-ras mutation. Conclusion CTNNB1 mutations seem to be of minor importance in sporadic colorectal cancer. The main differences in tumour and patient characteristics are found between groups of patients based on mismatch repair deficiency.

There has been a resurgence of interest in immunotherapies for cancer, particularly concerning the isolation of novel antigens which distinguish cancer cells from normal cells. Recently i t was demonstrated that the humoral immune response can be used to isolate novel tumour antigens. This involved the probing of a tumour specific cDNA expression library in h phage with autologous patient serum (serological cDNA screening). Using this method Sahin et al., (1%5)[1] have been able to identify new tumour antigens in melanoma, renal cell carcinoma, Hodgkin's disease and astrocytoma. We are applying serological cDNA screening to colorectal cancer (CRC) but are also extending this approach by allying it to phage display technology through the serological selection of phage displayed CRC cDNA libraries. Both approaches require the use of the patient's anti-tumour cell response to identify those tumour cDNA expression products that naturally evoke an IgG immune response, and as such may be interesting candidates for immunotherapy. We have made two colon tumour cDNA libraries in I-ZAP. The size of the libraries was greater than 106 recombinants with an average insert size of 1 kb, and with 1% positive GAPDH positive plaques. The quality of our libraries has also been tested by DNA probing for CEA which has shown approximately 0.022% CEA positive recombinants. One of our tumour cDNA h expression libraries has been screened using autologous paticnt serum. h phage recombinants were plated at the desired plaque density for screening and expression of the cDNA products [ 11. The library was seen to have a high proportion of IgG transcripts (0.1%) which are detected by the secondary antibody alone (antihuman IgG alkaline phosphatase conjugate) and complicate the screening procedure significantly. This high proportion of immunoglobulin transcripts in the cDNA library is most likely caused by high B-cell infiltration into the tumour. This in turn may be associated with a high humoral immune response. Serological cDNA screening has proven difficult due to the high proportion of IgG transcripts present and as such we are pursuing alternative approaches for the isolation of tumour antigens in CRC. We are using serological selection of cDNA libraries displayed on filamentous phage. The filamentous phage links the expressed and displayed protein product to the gene, cloned into the phage genome. This allows the selection and enrichment of those cDNA products that interact with a chosen immobilised, or labelled ligand, for example, in our case, autologous patient serum IgG. Selection from phage cDNA display has potential advantages over serological screening of cDNA libraries in I phage. (a) The gene product and its genetic information are physically linked through the display on thc surface of filamentous phage so retrieval of positive clones is much quicker and less labour intensive. (b) By using phage display we can enrich for specific genes via interactions between gene product and ligand. (c) The correct secretion and processing of the cDNA products is a pre-requisite for the display on filamentous phage. This means that phage displayed cDNA libraries will bias for cell surface tumour antigens (i.e naturally secreted antigens that can move through the E.coli secretion apparatus). (d) Proteins displayed on phage surfaces are likely to have an authentic three-dimensional structure so serum reactivity with conformational epitopes will operate in the selection procedure unlike in serological cDNA screening. (e) The conditions set for proteinlprotein interactions can be controlled during the selection procedure as all selections are done in vitro . The presence of stop codons in cDNA has suggested that direct display by fusion to the N-terminus of p3 by established methods is limited. As such an indirect display approach has been developed by Crameri and Suter (1994)[2] which involves the linking of cDNA libraries to the phage surface via the heterodimerisation of the Jun and Fos leucine zippers. A Jun-p3 fusion and a Fos-cDNA fusion are expressed in the same E.cofi cell and both products are exported to the periplasm where viral assembly occurs. The Jun-p3 fusion is incorporated into the M I 3 phage coat and the Fos-cDNA product can heterodimerise with the Jun leucine zipper such that it is displayed on the phage surface. This approach has been successfully applied to selection of allergenic proteins from a cDNA library prepared from Aspergillusfumigatus using serum from affected patients [2,3]. This vector (pJuFo) was made available by Dr. M. Suter for our own work. cDNA libraries have also been displayed on p6 [4] which connects the phage body to the host binding p3 and, unlike p3, it is thought to have a solvent exposed C-terminus available for the attachment of foreign proteins. We have constructed our own p6 display vectors, pSP6A, pSP6B and pSP6C which have a versatile multiple cloning site for the display of cDNA libraries in all three reading frames. We have displayed the CH3 domain of human IgG and alkaline phosphatase on p6 and compared the display efficiency with that of display on p3. Our results are in agreement with those of Jespers (1995)[4] in that p6 display efficiency is lower than p3 display (Manuscript in preparation). The two cDNA libraries that we have generated in h phage for display systems. We have cloned these libraries into both the

As a step towards understanding the complex differences between normal cells and cancer cells, we have used suppression subtractive hybridization (SSH) to generate a profile of genes overexpressed in primary colorectal cancer (CRC). From a 35¿ omitted¿000 clone SSH-cDNA repertoire, we have screened 400 random clones by reverse Northern blotting, of which 45 clones were scored as overexpressed in tumor compared to matched normal mucosa. Sequencing showed 37 different genes and of these, 16 genes corresponded to known genes in the public databases. Twelve genes, including Smad5 and Fls353, have previously been shown to be overexpressed in CRC. A series of known genes which have not previously been reported to be overexpressed in cancer were also recovered: Hsc70, PBEF, ribophorin II and Ese-3B. The remaining 21 genes have as yet no functional annotation. These results show that SSH in conjunction with high throughput screening provides a very efficient means to produce a broad profile of genes differentially expressed in cancer. Some of the genes identified may provide novel points of therapeutic intervention.