Acute WNT signalling activation perturbs differentiation within the adult stomach and rapidly leads to tumour formation

A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active β-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear β-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach

A fully human anti-Ep-CAM scFv-beta-glucuronidase fusion protein for selective chemotherapy with a glucuronide prodrug

Monoclonal antibodies against tumour-associated antigens could be useful to deliver enzymes selectively to the site of a tumour for activation of a non-toxic prodrug. A completely human fusion protein may be advantageous for repeated administration, as host immune responses may be avoided. We have constructed a fusion protein consisting of a human single chain Fv antibody, C28, against the epithelial cell adhesion molecule and the human enzyme β-glucuronidase. The sequences encoding C28 and human enzyme β-glucuronidase were joined by a sequence encoding a flexible linker, and were preceded by the IgGκ signal sequence for secretion of the fusion protein. A CHO cell line was engineered to secrete C28-β-glucuronidase fusion protein. Antibody specificity and enzyme activity were retained in the secreted fusion protein that had an apparent molecular mass of 100 kDa under denaturing conditions. The fusion protein was able to convert a non-toxic prodrug of doxorubicin, N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate to doxorubicin, resulting in cytotoxicity. A bystander effect was demonstrated, as doxorubicin was detected in all cells after N-[4-doxorubicin-N-carbonyl(oxymethyl)phenyl]-O-β-glucuronyl carbamate administration when only 10% of the cells expressed the fusion protein. This is the first fully human and functional fusion protein consisting of an scFv against epithelial cell adhesion molecule and human enzyme β-glucuronidase for future use in tumour-specific activation of a non-toxic glucuronide prodrug

Ratio of the Isolated Photon Cross Sections at \sqrt{s} = 630 and 1800 GeV

The inclusive cross section for production of isolated photons has been measured in \pbarp collisions at $\sqrt{s} = 630$ GeV with the \D0 detector at the Fermilab Tevatron Collider. The photons span a transverse energy ($E_T$) range from 7-49 GeV and have pseudorapidity $|\eta| < 2.5$. This measurement is combined with to previous \D0 result at $\sqrt{s} = 1800$ GeV to form a ratio of the cross sections. Comparison of next-to-leading order QCD with the measured cross section at 630 GeV and ratio of cross sections show satisfactory agreement in most of the $E_T$ range.Comment: 7 pages. Published in Phys. Rev. Lett. 87, 251805, (2001

Decreased expression of the mannose 6- phosphate/insulin-like growth factor-II receptor promotes growth of human breast cancer cells

BACKGROUND: Loss or mutation of the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) has been found in breast cancer. However, whether or not decreased levels of functional M6P/IGF2R directly contribute to the process of carcinogenesis needs to be further verified by functional studies. METHODS: In this study, using viral and ribozyme strategies we reduced the expression of M6P/IGF2R in human breast cancer cells and then examined the effect on growth and apoptosis of these cells. RESULTS: Our results showed that infection of MCF-7 cells with the adenovirus carrying a ribozyme targeted against the M6P/IGF2R mRNA dramatically reduced the level of transcripts and the functional activity of M6P/IGF2R in these cells. Accordingly, cells treated with the ribozyme exhibited a higher growth rate and a lower apoptotic index than control cells (infected with a control vector). Furthermore, decreased expression of M6P/IGF2R enhanced IGF-II-induced proliferation and reduced cell susceptibility to TNF-induced apoptosis. CONCLUSIONS: These results suggest that M6P/IGF2R functions as a growth suppressor and its loss or mutation may contribute to development and progression of cancer. This study also demonstrates that adenoviral delivery of the ribozyme provides a useful tool for investigating the role of M6P/IGF2R in regulation of cell growth

Expression of PPARδ in multistage carcinogenesis of the colorectum: implications of malignant cancer morphology

Whether peroxisome proliferator-activated receptor (PPAR) δ is a good target for the chemoprevention and/or treatment of colorectal cancer (CRC) remains controversial. Our goal was to examine PPARδ expression in multistage carcinogenesis of the colorectum and to assess the relevance of PPARδ in CRC. Immunohistochemical analysis indicated that PPARδ expression increased from normal mucosa to adenomatous polyps to CRC. In cancer tissues, the PPARδ protein was accumulated only in those cancer cells with highly malignant morphology, as represented by a large-sized nucleus, round-shaped nucleus, and presence of clear nucleoli. Interestingly, the cancer tissue often contained both PPARδ-positive and -negative areas, each retaining their respective specific morphological features. Moreover, this pattern persisted even when PPARδ-positive and -negative cells were aligned next to each other within a single cancer nest or gland and was present in the majority of CRC cases. Immunohistochemistry for Ki-67 proliferation marker showed no significant correlation between Ki-67 and PPARδ in CRC samples. Based on Western blot analysis and quantitative RT–PCR, high PPARδ protein expression correlated with high PPARδ mRNA levels. Peroxisome proliferator-activated receptor δ may have a supporting role in tumorigenesis, and the close association between PPARδ expression and malignant morphology of CRC cells suggests a pivotal role in cancer tissue

In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy

Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ΔRD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos

Antimetastatic activity of a cyclooxygenase-2 inhibitor

Cyclooxygenase-2 (COX-2) expression is increased in breast cancer and surgery has been shown to increase the growth of metastatic tumours. We investigated the effect of selective COX-2 inhibition on the growth of metastases in either an experimental metastasis model or following excision of a murine primary breast tumour. 50,000 4T1 mammary carcinoma cells were injected into the mammary fat pad of female BALB/c mice. When the mean TD reached 8+/-0.4 mm, tumours were excised and the mice were randomised into two groups (n=12 per group) to receive daily intraperitoneal injections of the selective COX-2 inhibitor, SC-236 or drug vehicle for 14 days. Alternatively, experimental metastases were established by tail-vein injection of 50,000 4T1 cells. Mice received either the selective COX-2 inhibitor, SC-236 or drug vehicle for 14 days (n=12 per group). SC-236 treatment significantly reduced tumour burden, the number and size of spontaneous metastases following primary tumour excision. SC-236 treatment also reduced tumour burden, the number and size of experimental metastases. Immunohistochemical staining demonstrated that COX-2 inhibition reduced microvessel density and increased apoptosis within both spontaneous and experimental metastases. These data clearly demonstrate that the selective COX-2 inhibitor, SC-236, has potent antimetastatic activity against both spontaneous metastases arising following primary tumour excision and experimental metastases.</p

Claudin-7 Is Frequently Overexpressed in Ovarian Cancer and Promotes Invasion

Background: Claudins are tight junction proteins that are involved in tight junction formation and function. Previous studies have shown that claudin-7 is frequently upregulated in epithelial ovarian cancer (EOC) along with claudin-3 and claudin-4. Here, we investigate in detail the expression patterns of claudin-7, as well as its possible functions in EOC. Methodology/Principal Findings: A total of 95 ovarian tissue samples (7 normal ovarian tissues, 65 serous carcinomas, 11 clear cell carcinomas, 8 endometrioid carcinomas and 4 mucinous carcinomas) were studied for claudin-7 expression. In real-time RT-PCR analysis, the gene for claudin-7, CLDN7, was found to be upregulated in all the tumor tissue samples studied. Similarly, immunohistochemical analysis and western blotting showed that claudin-7 protein was significantly overexpressed in the vast majority of EOCs. Small interfering RNA-mediated knockdown of claudin-7 in ovarian cancer cells led to significant changes in gene expression as measured by microarrays and validated by RT-PCR and immunoblotting. Analyses of the genes differentially expressed revealed that the genes altered in response to claudin-7 knockdown were associated with pathways implicated in various molecular and cellular functions such as cell cycle, cellular growth and proliferation, cell death, development, and cell movement. Through functional experiments in vitro, we found that both migration and invasion were altered in cells where CLDN7 had been knocked down or overexpressed. Interestingly, claudin-7 expression was associated with a net increase in invasion, but also with a decrease in migration

Comparison of Proliferation and Genomic Instability Responses to WRN Silencing in Hematopoietic HL60 and TK6 Cells

BACKGROUND: Werner syndrome (WS) results from defects in the RecQ helicase (WRN) and is characterized by premature aging and accelerated tumorigenesis. Contradictorily, WRN deficient human fibroblasts derived from WS patients show a characteristically slower cell proliferation rate, as do primary fibroblasts and human cancer cell lines with WRN depletion. Previous studies reported that WRN silencing in combination with deficiency in other genes led to significantly accelerated cellular proliferation and tumorigenesis. The aim of the present study was to examine the effects of silencing WRN in p53 deficient HL60 and p53 wild-type TK6 hematopoietic cells, in order to further the understanding of WRN-associated tumorigenesis. METHODOLOGY/PRINCIPAL FINDINGS: We found that silencing WRN accelerated the proliferation of HL60 cells and decreased the cell growth rate of TK6 cells. Loss of WRN increased DNA damage in both cell types as measured by COMET assay, but elicited different responses in each cell line. In HL60 cells, but not in TK6 cells, the loss of WRN led to significant increases in levels of phosphorylated RB and numbers of cells progressing from G1 phase to S phase as shown by cell cycle analysis. Moreover, WRN depletion in HL60 cells led to the hyper-activation of homologous recombination repair via up-regulation of RAD51 and BLM protein levels. This resulted in DNA damage disrepair, apparent by the increased frequencies of both spontaneous and chemically induced structural chromosomal aberrations and sister chromatid exchanges. CONCLUSIONS/SIGNIFICANCE: Together, our data suggest that the effects of WRN silencing on cell proliferation and genomic instability are modulated probably by other genetic factors, including p53, which might play a role in the carcinogenesis induced by WRN deficiency