RNA interferences in ES cells

The aim of this project was to develop methods for conditional induction of gene specific silencing in murine embryonic stem (mES) cells using RNA interference (RNAi). RNA interference (RNAi) is a process whereby double stranded (ds) RNA can reduce the expression of a gene by selective mRNA degradation. This thesis describes the investigation of several methods of inducing RNAi in mES cells. These included design and transfection of chemically synthesised small interfering (si) RNAs the transfection of vectors expressing short hairpin (sh) RNAs and construction of a vector that expresses hairpin dsRNA under tetracycline control. A specific reduction in GFP expression was observed by the transfection of siRNAs targeting either plasmid or endogenously expressed GFP. Transfections of two out of three Oct-4 siRNAs were shown to reduce the expression of Oct-4. mES cell lines were made that stably express shRNAs targeting Rex-1 and Laminin Bl. Analysis of gene expression by semi-quantitative RT-PCR showed knockdown of the target genes in some cell lines. Transfection of another vector expressing Oct-4 shRNA was shown to induce differentiation of mES cells as measured by an increase in cell size. Methods were developed for the cloning of an inverted-repeat of target genes (Oct-4, LMNA) into a tetracycline-inducible vector. The system was tested in a HEK 293 cell line expressing the tet-responsive transactivator protein. The hairpin dsRNA was shown to be expressed when doxycycline was added to the cells but no knockdown of LMNA was observed. The use of siRNAs and shRNAs to induce gene specific silencing in ES cells was shown. Although fast and efficient methods for the assaying of RNAi induced knockdown in cells were not demonstrated. Therefore the potential of RNAi as a high throughput system for establishing gene function in mES has yet to be realised

c-Src drives intestinal regeneration and transformation

The non‐receptor tyrosine kinase c‐Src, hereafter referred to as Src, is overexpressed or activated in multiple human malignancies. There has been much speculation about the functional role of Src in colorectal cancer (CRC), with Src amplification and potential activating mutations in up to 20% of the human tumours, although this has never been addressed due to multiple redundant family members. Here, we have used the adult <i>Drosophila</i> and mouse intestinal epithelium as paradigms to define a role for Src during tissue homeostasis, damage‐induced regeneration and hyperplasia. Through genetic gain and loss of function experiments, we demonstrate that Src is necessary and sufficient to drive intestinal stem cell (ISC) proliferation during tissue self‐renewal, regeneration and tumourigenesis. Surprisingly, Src plays a non‐redundant role in the mouse intestine, which cannot be substituted by the other family kinases Fyn and Yes. Mechanistically, we show that Src drives ISC proliferation through upregulation of EGFR and activation of Ras/MAPK and Stat3 signalling. Therefore, we demonstrate a novel essential role for Src in intestinal stem/progenitor cell proliferation and tumourigenesis initiation <i>in vivo.</i&gt

Large Cell Neuroendocrine Carcinoma of the Ampulla of Vater

Context Large cell neuroendocrine carcinomas of the ampulla of Vater are rare and confer a very poor prognosis despite aggressive therapy. There are few case reports of large cell neuroendocrine carcinomas of the ampulla of Vater in the literature and to date no studies have been done to establish optimal management. We describe a pooled case series from published reports of neuroendocrine carcinomas of the ampulla of Vater including a case which presented to our institution. Methods A narrative review was undertaken including all published English case reports of large cell neuroendocrine carcinomas of the ampulla of Vater. Our primary outcome was to determine the overall survival. Results Twenty cases of large cell neuroendocrine carcinomas of the ampulla of Vater were identified. Seventy-six percent of patients were reported to have died of disease with a mean survival of 11.8 months. Twenty percent of the tumours were associated with an adenoma. The approximate median survivals were 15 months for those with an associated adenoma and 11 months without. Conclusions This pooled analysis demonstrates both the rarity and poor prognosis of large cell neuroendocrine carcinomas of the ampulla of Vater. Although surgical resection is the mainstay of treatment, we review common adjuvant chemotherapy regimes. Prognosis may be improved when these tumours are associated with adenomas, however, further studies are needed.Image: Circumferential fleshy ulcerated tumour

The in vivo function of the p53 target gene TIGAR

The p53 tumour suppressor inhibits tumour development via various mechanisms such as apoptosis, inhibition of proliferation or the activation of senescence. Recently, several studies have indicated a novel role of p53 in the regulation of energy metabolism. Previously we have discovered TIGAR, a p53 target gene that acts as a fructose-2,6-bisphosphatase. TIGAR therefore can redirect glucose from the glycolytic pathway to the pentose phosphate pathway (PPP), which promotes NADPH production to generate reduced glutathione for protecting against ROS, and also ribose 5 phosphate production for nucleotide synthesis. In order to understand the function of TIGAR in vivo, we generated TIGAR deficient mice. We have determined a critical role of TIGAR in rapidly proliferating tissue, either for repair after damage or during tumor development

The pro-apoptotic K-Ras 4A proto-oncoprotein does not affect tumorigenesis in the ApcMin/+ mouse small intestine.

BACKGROUND: Alterations in gene splicing occur in human sporadic colorectal cancer (CRC) and may contribute to tumour progression. The K-ras proto-oncogene encodes two splice variants, K-ras 4A and 4B, and K-ras activating mutations which jointly affect both isoforms are prevalent in CRC. Past studies have established that splicing of both the K-ras oncogene and proto-oncogene is altered in CRC in favour of K-ras 4B. The present study addressed whether the K-Ras 4A proto-oncoprotein can suppress tumour development in the absence of its oncogenic allele, utilising the ApcMin/+ (Min) mouse that spontaneously develops intestinal tumours that do not harbour K-ras activating mutations, and the K-rastmDelta4A/tmDelta4A mouse that can express the K-ras 4B splice variant only. By this means tumorigenesis in the small intestine was compared between ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice that can, and cannot, express the K-ras 4A proto-oncoprotein respectively. METHODS: The relative levels of expression of the K-ras splice variants in normal small intestine and small intestinal tumours were quantified by real-time RT-qPCR analysis. Inbred (C57BL/6) ApcMin/+, K-ras+/+ and ApcMin/+, K-rastmDelta4A/tmDelta4A mice were generated and the genotypes confirmed by PCR analysis. Survival of stocks was compared by the Mantel-Haenszel test, and tumour number and area compared by Student's t-test in outwardly healthy mice at approximately 106 and 152 days of age. DNA sequencing of codons 12, 13 and 61 was performed to confirm the intestinal tumours did not harbour a K-ras activating mutation. RESULTS: The K-ras 4A transcript accounted for about 50% of K-ras expressed in the small intestine of both wild-type and Min mice. Tumours in the small intestine of Min mice showed increased levels of K-ras 4B transcript expression, but no appreciable change in K-ras 4A transcript levels. No K-ras activating mutations were detected in 27 intestinal tumours derived from Min and compound mutant Min mice. K-Ras 4A deficiency did not affect mouse survival, or tumour number, size or histopathology. CONCLUSION: The K-Ras 4A proto-oncoprotein does not exhibit tumour suppressor activity in the small intestine, even though the K-ras 4A/4B ratio is reduced in adenomas lacking K-ras activating mutations.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

E-cadherin can limit the transforming properties of activating β-catenin mutations

Wnt pathway deregulation is a common characteristic of many cancers. But only Colorectal Cancer predominantly harbours mutations in APC, whereas other cancer types (hepatocellular carcinoma, solid pseudopapillary tumours of pancreas) have activating mutations in β-catenin (CTNNB1). We have compared the dynamics and the potency of β-catenin mutations in vivo. Within the murine small intestine (SI), an activating mutation of β-catenin took much longer to achieve a Wnt deregulation and acquire a crypt-progenitor-cell (CPC) phenotype than Apc or Gsk3 loss. Within the colon, a single activating mutation of β-catenin was unable to drive Wnt deregulation or induce the CPC phenotype. This ability of β-catenin mutation to differentially transform the SI versus the colon correlated with significantly higher expression of the β-catenin binding partner E-cadherin. This increased expression is associated with a higher number of E-cadherin:β-catenin complexes at the membrane. Reduction of E-cadherin synergised with an activating mutation of β-catenin so there was now a rapid CPC phenotype within the colon and SI. Thus there is a threshold of β-catenin that is required to drive transformation and E-cadherin can act as a buffer to prevent β-catenin accumulation

RAL GTPases drive intestinal stem cell function and regeneration through internalization of WNT signalosomes

Ral GTPases are RAS effector molecules and by implication a potential therapeutic target for RAS mutant cancer. However, very little is known about their roles in stem cells and tissue homeostasis. Using Drosophila, we identified expression of RalA in intestinal stem cells (ISCs) and progenitor cells of the fly midgut. RalA was required within ISCs for efficient regeneration downstream of Wnt signaling. Within the murine intestine, genetic deletion of either mammalian ortholog, Rala or Ralb, reduced ISC function and Lgr5 positivity, drove hypersensitivity to Wnt inhibition, and impaired tissue regeneration following damage. Ablation of both genes resulted in rapid crypt death. Mechanistically, RALA and RALB were required for efficient internalization of the Wnt receptor Frizzled-7. Together, we identify a conserved role for RAL GTPases in the promotion of optimal Wnt signaling, which defines ISC number and regenerative potential

Loss of BCL9/9l suppresses Wnt driven tumourigenesis in models that recapitulate human cancer.

Different thresholds of Wnt signalling are thought to drive stem cell maintenance, regeneration, differentiation and cancer. However, the principle that oncogenic Wnt signalling could be specifically targeted remains controversial. Here we examine the requirement of BCL9/9l, constituents of the Wnt-enhanceosome, for intestinal transformation following loss of the tumour suppressor APC. Although required for Lgr5+ intestinal stem cells and regeneration, Bcl9/9l deletion has no impact upon normal intestinal homeostasis. Loss of BCL9/9l suppressed many features of acute APC loss and subsequent Wnt pathway deregulation in vivo. This resulted in a level of Wnt pathway activation that favoured tumour initiation in the proximal small intestine (SI) and blocked tumour growth in the colon. Furthermore, Bcl9/9l deletion completely abrogated β-catenin driven intestinal and hepatocellular transformation. We speculate these results support the just-right hypothesis of Wnt-driven tumour formation. Importantly, loss of BCL9/9l is particularly effective at blocking colonic tumourigenesis and mutations that most resemble those that occur in human cancer

Spectroscopy, MOST Photometry, and Interferometry of MWC 314: Is it an LBV or an interacting binary?

MWC 314 is a bright candidate luminous blue variable that resides in a fairly close binary system, with an orbital period of 60.753$\pm$0.003 d. We observed MWC 314 with a combination of optical spectroscopy, broad-band ground- and space-based photometry, as well as with long baseline, near-infrared interferometry. We have revised the single-lined spectroscopic orbit and explored the photometric variability. The orbital light curve displays two minima each orbit that can be partially explained in terms of the tidal distortion of the primary that occurs around the time of periastron. The emission lines in the system are often double-peaked and stationary in their kinematics, indicative of a circumbinary disc. We find that the stellar wind or circumbinary disc is partially resolved in the K\prime-band with the longest baselines of the CHARA Array. From this analysis, we provide a simple, qualitative model in an attempt to explain the observations. From the assumption of Roche Lobe overflow and tidal synchronisation at periastron, we estimate the component masses to be M1 $\approx 5$ M$_\odot$ and M2$\approx 15$ M$_\odot$, which indicates a mass of the LBV that is extremely low. In addition to the orbital modulation, we discovered two pulsational modes with the MOST satellite. These modes are easily supported by a low-mass hydrogen-poor star, but cannot be easily supported by a star with the parameters of an LBV. The combination of these results provides evidence that the primary star was likely never a normal LBV, but rather is the product of binary interactions. As such, this system presents opportunities for studying mass-transfer and binary evolution with many observational techniques.Comment: 26 pages, 7 figures, 5 tables, 2 appendices with 7 additional tables and 2 additional figures. Accepted for publication in MNRA

Intravital FRAP imaging using an E-cadherin-GFP mouse reveals disease- and drug-dependent dynamic regulation of cell-cell junctions in live tissue

E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments