Phospholipase C-gamma1 in tumor progression

The vast majority of cancer morbidity and mortality arises from tumor progression beyond the primary tumor site. Unfortunately, most therapies are not effective for advanced stage disease with regional extension or distant metastases. Thus, new treatments are needed to target rate limiting steps in tumor progression. The ability of cancers to invade and metastasize requires the acquisition of specific cell behaviors that enable the cell to escape from the localized site, breach the defined boundaries, reach a hospitable ectopic site and grow in this new locale. Recently, dysregulation of cell motility as stimulated by various extracellular factors has gained credence as a rate-limiting alteration in tumor progression in carcinomas and some other solid tumors. This has focused attention on initiators of signaling cascades that regulate tumor migration. In this effort, one molecule, phospholipase C-γ1 (PLCγ), has been shown to function as a key molecular switch. This revised version was published online in July 2006 with corrections to the Cover Date.     

The biology of the combretastatins as tumour vascular targeting agents

The tumour vasculature is an attractive target for therapy. Combretastatin A-4 (CA-4) and A-1 (CA-1) are tubulin binding agents, structurally related to colchicine, which induce vascular-mediated tumour necrosis in animal models. CA-1 and CA-4 were isolated from the African bush willow, Combretum caffrum, and several synthetic analogues are also now available, such as the Aventis Pharma compound, AVE8062. More soluble, phosphated, forms of CA-4 (CA-4-P) and CA-1 (CA-1-P) are commonly used for in vitro and in vivo studies. These are cleaved to the natural forms by endogenous phosphatases and are taken up into cells. The lead compound, CA-4-P, is currently in clinical trial as a tumour vascular targeting agent. In animal models, CA-4-P causes a prolonged and extensive shut-down of blood flow in established tumour blood vessels, with much less effect in normal tissues. This paper reviews the current understanding of the mechanism of action of the combretastatins and their therapeutic potential.     

Functional analysis of the actin-binding protein,tropomyosin 1, in neuroblastoma

Tropomyosin 1 (TM1) is downregulated in a number of transformed cell types, and exogenous expression of TM1 can restore actin organisation and reverse cellular transformation. We find that TM1 is also downregulated in human neuroblastoma cell lines, correlating with increasing malignancy. However, exogenous TM1 does not restore actin cytoskeleton organisation in neuroblastoma cells.     

Expression and subcellular localization of Ror tyrosine kinase receptors are developmentally regulated in cultured hippocampal neurons

Ror1 and Ror2 are two novel receptor tyrosine kinases that have been implicated in neuronal differentiation in Caenorhabditis elegans. As a first step toward elucidating their role in the mammalian brain, we analyzed their expression and localization patterns in hippocampal neurons. Our results showed that both receptors are expressed from early stages of development and that their protein levels peak during periods of active synapse formation. Immunocytochemical analysis indicated that Ror1 and Ror2 are highly concentrated in the growth cones of immature neurons and are present throughout the somatodendritic compartment of mature hippocampal cells. Further analysis indicated that they are present not only in the cell membrane but also in Triton- and saponin-insoluble fractions, suggesting that they may be associated with both the cytoskeleton and membrane-bound organelles. Taken collectively, our results suggest that Ror1 and Ror2 might play a role during early stages of development in mammalian central neurons.     

Skin fragility and hypohidrotic ectodermal dysplasia resulting from ablation of plakophilin 1

We report a 2-year-old boy with an unusual autosomal recessively inherited skin disease comprising trauma-induced skin fragility and congenital ectodermal dysplasia affecting hair, nails and sweat glands. Skin biopsy showed widening of intercellular spaces between keratinocytes and ultrastructural findings of small, poorly formed desmosomes with reduced connections to the keratin filament cytoskeleton. Immunohistochemical analysis revealed a complete absence of staining for the accessory desmosomal plaque protein plakophilin 1 (PKP1; band 6 protein). The affected individual was a compound heterozygote for null mutations on both alleles of the PKP1 gene. Both mutations occurred within the amino terminus of PKP1, the domain which normally binds the cytoskeletal keratin filament network to the cell membrane. Apart from its localization within desmosomal plaques, PKP1 may also be present within the cytoplasm and nucleus and has putative roles in signal transduction and regulation of gene activity. The clinicopathological observations in this patient demonstrate the relevance of PKP1 to desmosome formation, cutaneous cell-cell adhesion and epidermal development and demonstrate the specific manifestations of human functional knockout mutations in this gene.     

The cells of the rabbit meniscus: their arrangement, interrelationship, morphological variations and cytoarchitecture

Four major morphologically distinct classes of cells were identified within the adult rabbit meniscus using antibodies to cytoskeletal proteins. Two classes of cell were present in the fibrocartilage region of the meniscus. These meniscal cells exhibited long cellular processes that extended from the cell body. A third cell type found in the inner hyaline-like region of the meniscus had a rounded form and lacked projections. A fourth cell type with a fusiform shape and no cytoplasmic projections was found along the superficial regions of the meniscus. Using a monoclonal antibody to connexin 43, numerous gap junctions were observed in the fibrocartilage region, whereas none were seen in cells either from the hyaline-like or the superficial zones of the meniscus. The majority of the cells within the meniscus exhibited other specific features such as primary cilia and 2 centrosomes. The placement of the meniscal cell subtypes as well as their morphology and architecture support the supposition that their specific characteristics underlie the ability of the meniscus to respond to different types of environmental mechanical loads.     

Eccentric exercise‐induced injuries to contractile and cytoskeletal muscle fibre components

Exercise involving lengthening of an activated muscle can cause injury. Recent reports documented the mechanics of exercise‐induced muscle injury as well as physiological and cellular events and manifestations of injury. Loss of the cytoskeletal protein desmin and loss of cellular integrity as evidenced by sarcolemmal damage occur early during heavy eccentric exercise. These studies indicate that the earliest events in muscle injury are mechanical in nature, while later events indicate that it may be more appropriate to conclude that intense exercise initiates a muscle remodeling process. We conclude that muscle injury after eccentric exercise is differently severe in muscles with different architecture, is fibre type‐specific, primarily because of fibre strain in the acute phase, and is exacerbated by inflammation after the initial injury.     

Alterations in neurofilaments associated with reactive brain changes and axonal sprouting following acute physical injury to the rat neocortex

In order to study the changes in axons related to acute localized physical trauma, a 25 gauge needle was inserted into the somatosensory cortex of anaesthetized adult rats. Animals were examined over 11 time points, from 30 min to 14 days postinjury. Initially, the central needle tract was surrounded by 'reactive' abnormal axons characterized by their bulb- or ring-like immunoreactivity for neurofila ments. Quantification demonstrated that these structures reached a peak density at 24 h postinjury, followed by a gradual decrease over 2 weeks. By 5 days postinjury, long axons showing high levels of neurofilament labelling were localized to the lesion area, either aligned parallel to the tract edges or extending into the bridge of tissue forming between the tract edges. Double-labelling demonstrated a close association between sprouting axons and ferritin-labelled microglia. Immunolabelling for GAP43 also demonstrated the presence of sprouting axons within this tissue bridge. Ultrastuctural examination showed that sprouting axons contained a high density of neurofilaments, with a leading edge lacking these filaments. Injury to the adult neocortex is associated with reactive and sprouting changes within axons, coordinated with the proliferation of microglia and wound healing. These data also support a role for neurofilaments in axonal sprouting following brain injury.     

The role of the membrane cytoskeleton cross-linker ezrin in medulloblastoma cells

Medulloblastoma is a highly malignant brain tumor that occurs predominantly in children. The molecular pathogenesis of medulloblastoma is under investigation. Previously, we used complementary DNA micro-array analysis to compare patterns of gene expression in medulloblastoma samples versus normal cerebellum. The cytoskeletal protein ezrin was found to be overexpressed in medulloblastoma compared with normal cerebellum, an observation that was further validated by immunohistochemistry and real-time PCR analysis. To assess the role of ezrin in medulloblastoma, we studied ezrin’s role in medulloblastoma migration, invasion, and adhesion. Western blotting and immunofluorescence showed high expression of ezrin in four medulloblastoma cell lines, and ezrin was primarily localized to filopodia. Ezrinspecific small interfering RNA suppressed the formation of filopodia and in vitro migration, invasion, and adhesion. We also used a stably transfected medulloblastoma cell line to study the effect of ezrin overexpression. We showed that high expression of ezrin promotes filopodia formation and in vitro invasion. Finally, athymic mice implanted with ezrin-overexpressing DAOY medulloblastoma cell clones in the cerebellum showed shortened survival compared with controls. These findings suggest that, in addition to other cytoskeletal proteins, ezrin plays an important role in medulloblastoma adhesion, migration, and invasion.