Dietetics, Nutrition and Biological Sciences
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Item Control of cell cycle gene expression in bone development and during c-Fos-induced osteosarcoma formation.(Wiley, 1998) Sunters, A.; McCluskey, Jane T.; Grigoriadis, A. E.We have used c-Fos transgenic mice which develop osteosarcomas to determine the expression patterns of cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs) in different bone cell populations in order to define the potential mechanisms of c-Fos transformation. Immunohistochemical analysis in embryonic and early postnatal bone demonstrated that cyclin E and its kinase partner CDK2 were expressed specifically in bone-forming osteoblasts. Cyclin D1 expression was absent despite high levels of CDK4 and CDK6, and the CKI p27 was expressed in chondrocytes, osteoclasts, and at lower levels in osteoblasts. Following activation of the c-fos transgene in vivo and before overt tumor formation, cyclin D1 expression increased dramatically and was colocalized with exogenous c-Fos protein specifically in osteoblasts and chondrocytes, but not in osteoclasts. Prolonged activation of c-Fos resulted in osteosarcoma formation wherein the levels of cyclin D1, cyclin E, and CDKs 2, 4, and 6 were high in a wide spectrum of malignant cell types, especially in transformed osteoblasts. The CKI p27 was expressed at very high levels in bone-resorbing osteoclasts, and to a lesser extent in chondrocytes and osteoblasts. These in vivo observations suggest that cyclin D1 may be a target for c-Fos action and that elevation of cyclin D1 in osteoblasts which already express cyclin E/CDK2 and the cyclin D1 partners CDKs-4 and 6, may predispose cells to uncontrolled cell growth leading to osteosarcoma development. This study implicates altered cell cycle control as a potential mechanism through which c-Fos causes osteoblast transformation and bone tumor formation.Item Perfect wound healing in the keratin 8 deficient mouse embryo.(Wiley, 1996) Brock, J.; McCluskey, Jane T.; Baribault, H.; Martin, P.It is generally believed that the strength and structural integrity of both adult and embryonic epithelia comes, at least in part, from their internal cytoskeletal network of keratin filaments and associated cell:cell junctions. Indeed, recent keratin depletion experiments in Xenopus suggest that the capacity of embryonic epithelia to undergo natural morphogenetic movements such as gastrulation, or artificially triggered epithelial movements such as wound closure, are severely compromised in the absence of the predominant embryonic keratin, K8 [Torpey et al., 1992: Nature 357:413-415; Klymkowsky et al., 1992: Proc. Natl. Acad. Sci. USA 89:8736-8740]. These experiments contrast with studies of genetically K8 deficient mouse embryos which undergo gastrulation quite normally and, dependent upon background strain, can survive until beyond birth [Baribault et al., 1993: Genes Dev. 7:1191-1202; Baribault et al., 1994: Genes Dev. 8:2964-2973], but to date no wound healing investigations have been carried out on mK8-mice. In this article, we report our studies of healing in embryonic day 11.5 mouse embryos, wounded by amputation of the hindlimb bud and then cultured in roller bottles. In wild-type embryos, wound closure puts severe strain on the embryonic epidermis since it is under tension and gapes immediately upon wounding; subsequently, epithelial cells tug on one another by means of an actin purse-string in order to close the defect. Even given these extremely challenging conditions, we show here that the mK8- epidermis performs no differently from wild-type epidermis, assembling an actin purse-string in the wound marginal cells and closing the wound with identical timecourse to its wild-type counterpart.Item Analysis of the Tissue Movements of Embryonic Wound Healing-DiI Studies in the Limb Bud Stage Mouse Embryo(Elsevier, 1995-07) McCluskey, Jane T.; Martin, PaulThe tissue movements of epithelial spreading and mesenchymal contraction play key roles in many aspects of embryonic morphogenesis. One way of studying these movements in a controlled manner is to make an excisional skin wound to an embryo and watch the wound heal. In this paper we report our studies of healing of a simple excisional lesion made to the limb bud stage mouse embryo. The wounded, living embryo is cultured in a roller bottle; under such conditions the wound heals with a highly reproducible time course and is completely closed by 24 hr. During the healing period the environment bathing the wound can be simply manipulated by adding drugs or factors to the culture medium. We have used DiI to label mesenchymal cells exposed at the margin of the initial wound and, by following their fate and measuring the area of mesenchyme remaining exposed at various time points during the healing process, we have quantified both the extent of mesenchymal contraction and the extent of reepithelialisation by movement of epidermis over mesenchyme. We show that the two types of tissue movement contribute almost equally (50:50) to the total wound closure rate. We have gone on to investigate the cell machinery underlying these processes. In adult wounds the epidermis migrates by means of lamellipodial crawling, but we show that reepithelialisation in the embryo is achieved instead by purse-string contraction of a cable of filamentous actin which assembles in the basal layer of cells at the free edge of the epidermis. Addition of cytochalasin D to the culture medium blocks formation of this actin cable and leads to failure of reepithelialisation. Contraction of adult wound connective tissue appears to be driven by conversion of dermal fibroblasts into a specialist smooth muscle-like fibroblast, the myofibroblast. However, using an antibody recognising the alpha-isoform of smooth muscle actin and specific for smooth muscle cells and myofibroblasts, we show that a similar conversion into myofibroblasts does not occur at any stage during the embryonic wound healing process. These observations indicate that both of the tissue movements of embryonic wound healing utilise cell machinery fundamentally different from that driving the analogous tissue movements of adult healing.Item Growth factors and wound healing(JAI Press, 1996) Martin, P.; McCluskey, Jane T.; Mallucci, P.; Nodder, S.; Bondy, C.; LeRoith, D.Item Repair of excisional wounds in the embryo(Nature Publishing Group, 1994) Martin, Paul; Nobes, Catherine; McCluskey, Jane T.; Lewis, JulianWound healing in the embryo, just as in the adult, comprises two tissue movements: re-epithelialisation and connective-tissue contraction. In this brief review we describe our recent studies of these two movements in both chick and rodent embryo model systems. In the chick we have evidence that the embryonic wound epidermis is drawn forwards by contraction of an actin pursestring extending around the circumference of the wound, rather than by lamellipodial crawling as in adult healing. Significant connective-tissue contraction also occurs. In the rat and mouse embryo we have examined expression of transcription factors and growth factors at the wound edge. We discuss our observations that the immediate-early gene c-fos and the growth factor transforming growth factor beta-1 are rapidly induced at the embryonic wound margin, and the possibility that these signals may trigger proliferation of wound edge cells and contraction of the exposed wound mesenchyme.Item Growth factors and cutaneous wound repair.(1992) Martin, P.; Hopkinson-Woolley, J.; McCluskey, Jane T.The healing of an adult skin lesion is a well studied but complex affair of some considerable clinical interest. Endogenous growth factors, including the EGF, FGF, PDGF and TGF beta families, are released at the wound site and presumed to be a necessary part of the natural wound healing machinery. Moreover, members of each of these families have been shown to enhance healing if added exogenously to a wound site. In this review we shall briefly discuss what is known about the mechanics and cell biology of adult wound healing, describe the normal cellular source of growth factors during the healing process and, with reference to their known capacities in tissue culture, speculate as to how particular growth factors might be able to enhance healing.Item Clinical implications of embryonic wound healing(Mark Allen Group, 1993) Hopkinson-Woolley, J.; McCluskey, Jane T.; Lewis, J.; Martin, P.An examination of research on the healing abilities of animal embryos and a consideration of its implications for human adult wound healingItem A study of wound healing in the E11.5 mouse embryo by light and electron microscopy(Elsevier, 1993-04) McCluskey, Jane T.; Hopkinson-Woolley, James; Luke, Babara; Martin, PaulIn this paper we report our light and electron microscopic studies of the healing of a simple excisional lesion to the E11.5 mouse embryo hindlimb. The wounded living embryo is cultured in a roller bottle and under such conditions the lesion is completely re-covered with epithelium by 24 hr. We discuss how our studies of such a simple wound healing model may offer insight into the mechanisms of tissue repair generally.