The APC tumour suppressor gene is mutated in most colon cancers. A major role of APC is the downregulation of the β-catenin/T-cell factor (Tcf)/lymphoid enhancer factor (LEF) signalling pathway; however, there are also suggestions that it plays a role in the organization of the cytoskeleton, and in cell adhesion and migration. For the first time, we have achieved stable expression of wild-type APC in SW480 colon cancer cells, which normally express a truncated form of APC. The ectopically expressed APC is functional, and results in the translocation of β-catenin from the nucleus and cytoplasm to the cell periphery, and reduces β-catenin/Tcf/LEF transcriptional signalling. E-cadherin is also translocated to the cell membrane, where it forms functional adherens junctions. Total cellular levels of E-cadherin are increased in the SW480APC cells and the altered charge distribution in the presence of full-length APC suggests that APC is involved in post-translational regulation of E-cadherin localization. Changes in the location of adherens junction proteins are associated with tighter cell-cell adhesion in SW480APC cells, with consequent changes in cell morphology, the actin cytoskeleton and cell migration in a wound assay. SW480APC cells have a reduced proliferation rate, a reduced ability to form colonies in soft agar and do not grow tumours in a xenograft mouse tumour model. By regulating the intracellular transport of junctional proteins, we propose that APC plays a role in cell adhesion in addition to its known role in β-catenin transcriptional signalling.

The cDNA for a human Class II phosphoinositide 3-kinase (PI 3-kinase C2␤) with a C2 domain was cloned from a U937 monocyte cDNA library and the enzyme expressed in mammalian and insect cells. Like other Class II PI 3-kinases in vitro, PI 3-kinase C2␤ utilizes phosphatidylinositol (PI) and PI 4-monophosphate but not PI 4,5-biphosphate as substrates in the presence of Mg 2؉ . Remarkably, and unlike other PI 3-kinases, the enzyme can use either Mg-ATP or Ca-ATP to generate PI 3-monophosphate. PI 3-kinase C2␤, like the Class I PI 3-kinases, but unlike PI 3-kinase C2␣, is sensitive to low nanomolar levels of the inhibitor wortmannin. The enzyme is not regulated by the small GTP-binding protein Ras. The C2 domain of the enzyme bound anionic phospholipids such as PI and phosphatidylserine in vitro, but did not co-operatively bind Ca 2؉ and phospholipids. Deletion of the C2 domain increased the lipid kinase activity suggesting that it functions as a negative regulator of the catalytic domain. Although presently it is not known whether PI 3-kinase C2␤ is regulated by Ca 2؉ in vivo, our results suggest a novel role for Ca 2؉ ions in phosphate transfer reactions.The cell, through diverse surface receptors with unique binding specificities, can sense bound signal molecules and transduce responses that regulate its physiology. It seems clear that most cell surface receptors activate a phosphoinositide 3-kinase 1 as part of the signal transduction cascade leading to the formation of phosphoinositides with 3Ј-phosphate groups (1, 2). The diversity of physiological events associated with increased PI 3-kinase activity is evident from reports of enzyme activation in: processes such as cell proliferation and transformation (3-5), events linked to insulin action, including alterations in glucose transport (6), the effects of growth factors on cell shape and motility (7), T cell signaling (8, 9) and apoptosis (10, 11). The activation of PI 3-kinase in this array of receptor-triggered processes suggests that 3Ј-phosphoinositides have a role as second messengers. At least three 3Ј-phosphoinositides are produced in cells: PI(3)P, PI(3,4)P 2 , and PI(3,4,5)P 3 . Receptor-triggered signals have been shown to activate PI 3-kinases and generate PI(3,4)P 2 and PI(3,4,5)P 3 (12, 13). PI(3)P can also be detected in cells but its production is not regulated by external signals (12, 13). PI(3)P and PI(3,4)P 2 can also be generated from PI(3,4)P 2 and PI(3,4,5)P 3 through the action of phosphoinositide phosphatases which could be regulated by distinct mechanisms to those of the phosphoinositide kinases (14). Through many studies involving the purification and molecular characterization of PI 3-kinases, a family of enzymes has been defined, which can be divided into three classes, whose members have diverse substrate specificity and distinct control mechanisms (15, 16). The Class I PI 3-kinases, which can be subdivided into IA and IB, are known to be activated by receptors. Although they can phosphorylate PI, PI(4)P, and PI(4,5)P 2 in vitro, t...

Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters measured in this report.