Characterizing system performance in total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRF-M) has become an increasingly popular tool to study events in close proximity to the cell cortex, such as cell adhesion (Axelrod, J Cell Biol 89:141-145, 1981; Gingell et al., J Cell Biol 100:1334-1338, 1985; Patel et al., J Cell Sci 121:1159-1164, 2008), actin (Bretschneider et al.

K-Ras and β-catenin mutations cooperate with Fgfr3 mutations in mice to promote tumorigenesis in the skin and lung, but not in the bladder.

The human fibroblast growth factor receptor 3 (FGFR3) gene is frequently mutated in superficial urothelial cell carcinoma (UCC). To test the functional significance of FGFR3 activating mutations as a 'driver' of UCC, we targeted the expression of mutated Fgfr3 to the murine urothelium using Cre-loxP recombination driven by the uroplakin II promoter. The introduction of the Fgfr3 mutations resulted in no obvious effect on tumorigenesis up to 18 months of age.

Wnt signalling and its role in stem cell-driven intestinal regeneration and hyperplasia.

Adult stem cells are crucial for normal tissue homeostasis and regeneration upon damage. Deregulated stem cell proliferation and/or differentiation have been linked to the formation and progression of tumours. Due to its high regenerative potential, the adult intestinal epithelium is an excellent system to study the mechanisms that underpin regeneration and transformation.

Ras mutation cooperates with β-catenin activation to drive bladder tumourigenesis.

Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels.

K-Ras mediated murine epidermal tumorigenesis is dependent upon and associated with elevated Rac1 activity.

A common goal for potential cancer therapies is the identification of differences in protein expression or activity that would allow for the selective targeting of tumor vs. normal cells. The Ras proto-oncogene family (K-Ras, H-Ras and N-Ras) are amongst the most frequently mutated genes in human cancers.

Structure-activity relationship and multidrug resistance study of new S-trityl-L-cysteine derivatives as inhibitors of Eg5.

The mitotic spindle is a validated target for cancer chemotherapy. Drugs such as taxanes and vinca alkaloids specifically target microtubules and cause the mitotic spindle to collapse. However, toxicity and resistance are problems associated with these drugs.

Metabolic regulation by p53.

We are increasingly aware that cellular metabolism plays a vital role in diseases such as cancer, and that p53 is an important regulator of metabolic pathways. By transcriptional activation and other means, p53 is able to contribute to the regulation of glycolysis, oxidative phosphorylation, glutaminolysis, insulin sensitivity, nucleotide biosynthesis, mitochondrial integrity, fatty acid oxidation, antioxidant response, autophagy and mTOR signalling. The ability to positively and negatively regulate many of these pathways, combined with feedback signalling from these pathways to p53, demonstrates the reciprocal and flexible nature of the regulation, facilitating a diverse range of responses to metabolic stress.

Inborn and acquired metabolic defects in cancer.

The observation that altered metabolism is the fundamental cause of cancer was made by Otto Warburg nearly a century ago. However, the subsequent identification of oncogenes and tumor suppressor genes has displaced Warburg's theory pointing towards genetic aberrations as the underlining cause of cancer. Nevertheless, in the last decade, cancer-associated mutations have been identified in genes coding for tricarboxylic acid cycle (TCA cycle, also known as Krebs cycle) and closely related enzymes that have essential roles in cellular metabolism.

Efficient Wnt mediated intestinal hyperproliferation requires the cyclin D2-CDK4/6 complex.

Inactivation of the gene encoding the adenomatous polyposis coli (APC) tumour suppressor protein is recognized as the key early event in the development of colorectal cancers (CRC). Apc loss leads to nuclear localization of beta-catenin and constitutive activity of the beta-catenin-Tcf4 transcription complex. This complex drives the expression of genes involved in cell cycle progression such as c-Myc and cyclin D2.

Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53.

The ability to observe changes in molecular behavior during cancer cell invasion in vivo remains a major challenge to our understanding of the metastatic process. Here, we demonstrate for the first time, an analysis of RhoA activity at a subcellular level using FLIM-FRET (fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer) imaging in a live animal model of pancreatic cancer. In invasive mouse pancreatic ductal adenocarcinoma (PDAC) cells driven by mutant p53 (p53(R172H)), we observed a discrete fraction of high RhoA activity at both the leading edge and rear of cells in vivo which was absent in two-dimensional in vitro cultures.

GRP78 up-regulation is associated with androgen receptor status, Hsp70-Hsp90 client proteins and castrate-resistant prostate cancer.

GRP78/BiP is a key member of the molecular chaperone heat shock protein (Hsp) 70 family. It has a critical role in prostate cancer (PC) including Pten loss-driven carcinogenesis, but the molecular basis of this remains unclear. We investigated the effect of GRP78 and its putative client proteins, including androgen receptor (AR) in clinical PC.

p53-mediated transcriptional regulation and activation of the actin cytoskeleton regulatory RhoC to LIMK2 signaling pathway promotes cell survival.

The central arbiter of cell fate in response to DNA damage is p53, which regulates the expression of genes involved in cell cycle arrest, survival and apoptosis. Although many responses initiated by DNA damage have been characterized, the role of actin cytoskeleton regulators is largely unknown. We now show that RhoC and LIM kinase 2 (LIMK2) are direct p53 target genes induced by genotoxic agents.

BID is cleaved by caspase-8 within a native complex on the mitochondrial membrane.

Caspase-8 stably inserts into the mitochondrial outer membrane during extrinsic apoptosis. Inhibition of caspase-8 enrichment on the mitochondria impairs caspase-8 activation and prevents apoptosis. However, the function of active caspase-8 on the mitochondrial membrane remains unknown.

Cytoplasmic ASPP1 inhibits apoptosis through the control of YAP.

The ASPP (apoptosis-stimulating protein of p53) family of proteins can function in the nucleus to modulate the transcriptional activity of p53, with ASPP1 and ASPP2 contributing to the expression of apoptotic target genes. In this study, we describe a new function for cytoplasmic ASPP1 in controlling YAP (Yes-associated protein)/TAZ. ASPP1 can inhibit the interaction of YAP with LATS1 (large tumor suppressor 1), a kinase that phosphorylates YAP/TAZ and promotes cytoplasmic sequestration and protein degradation.

Use of the parameterised finite element method to robustly and efficiently evolve the edge of a moving cell.

In this paper we present a computational tool that enables the simulation of mathematical models of cell migration and chemotaxis on an evolving cell membrane. Recent models require the numerical solution of systems of reaction-diffusion equations on the evolving cell membrane and then the solution state is used to drive the evolution of the cell edge. Previous work involved moving the cell edge using a level set method (LSM).

The dark side of fly TNF: an ancient developmental proof reading mechanism turned into tumor promoter.

The fruit fly Drosophila is an important model for biological research; however, due to its relatively short lifespan its relevance in cancer research is often questioned. Nevertheless, among many other intriguing Drosophila models, scribble group mutants provided early evidence for the existence of tumor suppressor genes and their importance in mammalian systems is beginning to emerge. In this review, I discuss recent advances in our understanding of the phenotypes of scrib group mutants, in which the activation of JNK signaling plays a crucial role.

LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.

LIM kinases 1 and 2 (LIMK1/2) are centrally positioned regulators of actin cytoskeleton dynamics. Using siRNA-mediated knockdown or a novel small molecule inhibitor, we show LIMK is required for path generation by leading tumor cells and nontumor stromal cells during collective tumor cell invasion. LIMK inhibition lowers cofilin phosphorylation, F-actin levels, serum response factor transcriptional activity and collagen contraction, and reduces invasion in three-dimensional invasion assays.

β-Catenin activation synergizes with PTEN loss to cause bladder cancer formation.

Although deregulation of the Wnt signalling pathway has been implicated in urothelial cell carcinoma (UCC), the functional significance is unknown. To test its importance, we have targeted expression of an activated form of β-catenin to the urothelium of transgenic mice using Cre-Lox technology (UroIICRE(+) β-catenin(exon3/+)). Expression of this activated form of β-catenin led to the formation of localized hyperproliferative lesions by 3 months, which did not progress to malignancy.

p53, ROS and senescence in the control of aging.

In addition to its function as a tumour suppressor, p53 is also involved in an increasing number of pathology associated with aging. Several activities of p53 appear contribute to its role in aging; one function that might be particularly relevant in this context is the regulation of senescence. The control of ROS and senescence by p53 may help to explain how p53 can function to both restrain and promote aging.

Fascin: Invasive filopodia promoting metastasis.

Fascin is an evolutionarily conserved actin bundling protein that localizes to microspikes, filopodia and actin-based protrusions underneath the plasma membrane. fascin has received a lot of attention among cytoskeletal proteins because multiple clinical studies have implicated its expression in cancer progression and metastasis. this may be because fascin is not normally expressed in epithelial tissues and when it is upregulated as a part of a program of cancer cell epithelial to mesenchymal progression it confers special motility and invasion properties on cancer cells.

Focal adhesion kinase is required for intestinal regeneration and tumorigenesis downstream of Wnt/c-Myc signaling.

The intestinal epithelium has a remarkable capacity to regenerate after injury and DNA damage. Here, we show that the integrin effector protein Focal Adhesion Kinase (FAK) is dispensable for normal intestinal homeostasis and DNA damage signaling, but is essential for intestinal regeneration following DNA damage. Given Wnt/c-Myc signaling is activated following intestinal regeneration, we investigated the functional importance of FAK following deletion of the Apc tumor suppressor protein within the intestinal epithelium.

Dying alone: a tale of rho.

In this issue of Cell Stem Cell, Chen et al. (2010) and Ohgushi et al. (2010) report that Rho-mediated actomyosin contractility underlies the proclivity of human embryonic stem cells to undergo apoptosis upon dissociation or during low-density propagation, a property that challenges their in vitro isolation and manipulation.

Akt/PKB suppresses DNA damage processing and checkpoint activation in late G2.

Using chemical genetics to reversibly inhibit Cdk1, we find that cells arrested in late G2 are unable to delay mitotic entry after irradiation. Late G2 cells detect DNA damage lesions and form gamma-H2AX foci but fail to activate Chk1. This reflects a lack of DNA double-strand break processing because late G2 cells fail to recruit RPA (replication protein A), ATR (ataxia telangiectasia and Rad3 related), Rad51, or CtIP (C-terminal interacting protein) to sites of radiation-induced damage, events essential for both checkpoint activation and initiation of DNA repair by homologous recombination.

PLP/DM20 expression and turnover in a transgenic mouse model of Pelizaeus-Merzbacher disease.

The most common cause of Pelizaeus-Merzbacher (PMD) is due to duplication of the PLP1 gene but it is unclear how increased gene dosage affects PLP turnover and causes dysmyelination. We have studied the dynamics of PLP/DM20 in a transgenic mouse model of PMD with increased gene dosage of the proteolipid protein gene (Plp1). The turnover of PLP/DM20 were investigated using an ex-vivo brain slice system and cultured oligodendrocytes.