M. Leigh Ackland

Epithelial-mesenchymal and mesenchymal : Epithelial transitions in carcinoma progression

Like a set of bookends, cellular, molecular, and genetic changes of the beginnings of life mirror those of one of the most common cause of death—metastatic cancer. Epithelial to mesenchymal transition (EMT) is an important change in cell phenotype which allows the escape of epithelial cells from the structural constraints imposed by tissue architecture, and was first recognized by Elizabeth Hay in the early to mid 1980s to be a central process in early embryonic morphogenesis. Reversals of these changes, termed mesenchymal to epithelial transitions (METs), also occur and are important in tissue construction in normal development. Over the last decade, evidence has mounted for EMT as the means through which solid tissue epithelial cancers invade and metastasize. However, demonstrating this potentially rapid and transient process in vivo has proven difficult and data connecting the relevance of this process to tumor progression is still somewhat limited and controversial. Evidence for an important role of MET in the development of clinically overt metastases is starting to accumulate, and model systems have been developed. This review details recent advances in the knowledge of EMT as it occurs in breast development and carcinoma and prostate cancer progression, and highlights the role that MET plays in cancer metastasis. Finally, perspectives from a clinical and translational viewpoint are discussed. J. Cell. Physiol. 213: 374–383, 2007.

Multicellular spheroids in ovarian cancer metastases: Biology and pathology.

Epithelial ovarian cancer (EOC) has a relatively high mortality rate ( approximately 55%). One of the presiding causes is that the current chemotherapeutic regimes are unable to achieve sustained remission, despite frequently producing a positive response at first treatment. One of the reasons that EOC is difficult to treat is that the mechanism of dissemination is unusual. EOC dissemination characteristically involves local invasion of pelvic and abdominal organs. Unlike many epithelial cancers, initial dissemination rarely requires the vasculature, although the vasculature is often implicated in the advanced stages of disease. Recently, it has become apparent that aggregates of malignant cells (spheroids) contained within malignant ascites represent a significant impediment to efficacious treatment of late stage EOC. In vivo, spheroids are present in the malignant ascites of EOC patients, while in vitro cultured spheroids are capable of tumorgenesis in vivo and display a reduced response to chemotherapeutic drugs when compared to monolayers. A major problem associated with the current generation of chemotherapy agents is that they do not address the anchorage- and vascular-independent growth conditions associated with a 3-dimensional structure that has formed and/or grown in suspension. Thus, spheroid formation may represent a key component of platinum/taxane-sensitive recurrence. If this is correct, a better understanding of spheroid biology may contribute to the identification of new treatment opportunities for the sustained treatment of metastatic EOC. This review article outlines the key biological features of spheroids, specifically discussing their role in EOC dissemination and chemo-response as well as providing insights into spheroid functionality.

Intracellular zinc homeostasis in leukocyte subsets is regulated by different expression of zinc exporters ZnT‐1 to ZnT‐9

Intracellular zinc homeostasis is strictly regulated by zinc binding proteins and zinc transporters. In the present study, we quantified in a first global view the expression of all characterized human zinc exporters (hZnT‐1‐9) in different leukocyte subsets in response to zinc supplementation and depletion and analyzed their influence on alterations in the intracellular zinc concentration. We found that hZnT‐1 is the most regulated zinc exporter. Furthermore, we discovered that hZnT‐4 is localized in the plasma membrane similar to hZnT‐1. hZnT‐4 is most highly expressed in Molt‐4, up‐regulated after treatment with PHA and is responsible for the measured decrease of intracellular zinc content after high zinc exposure. In addition, we found that hZnT‐5, hZnT‐6, and hZnT‐7 in Raji as well as hZnT‐6 and hZnT‐7 in THP‐1 are up‐regulated in response to cellular zinc depletion. Those zinc exporters are all localized in the Golgi network, and this type of regulation explains the observed zinc increase in both cell types after up‐regulation of their expression during zinc deficiency and, subsequently, high zinc exposure. Furthermore, we detected, for the first time, the expression of hZnT‐8 in peripheral blood lymphocytes, which varied strongly between individuals. While hZnT‐2 was not detectable, hZnT‐3 and hZnT‐9 were expressed at low levels. Further on, the amount of expression was higher in primary cells than in cell lines. These data provide insight into the regulation of intracellular zinc homeostasis in cells of the immune system and may explain the variable effects of zinc deficiency on different leukocyte subsets.

Zinc deficiency and its inherited disorders - A review

Zinc is an essential trace element required by all living organisms because of its critical roles both as a structural component of proteins and as a cofactor in enzyme catalysis. The importance of zinc in human metabolism is illustrated by the effects of zinc deficiency, which include a diminished immune response, reduced healing and neurological disorders. Furthermore, nutritional zinc deficiency can be fatal in newborn or growing animals. While zinc deficiency is commonly caused by dietary factors, several inherited defects of zinc deficiency have been identified. Acrodermatitis enteropathica is the most commonly described inherited condition found in humans. In several of the few cases that have been reported, this disorder is associated with mutations in the hZIP4 gene, a member of the SLC39 family, whose members encode membranebound putative zinc transporters. Mutations in other members of this family or in different genes may account for other cases of acrodermatitis in which defects in hZIP4 have not been detected. Another inherited form of zinc deficiency occurs in the lethal milk mouse, where a mutation in ZnT4 gene, a member of the SLC30 family of transmembrane proteins results in impaired secretion of zinc into milk from the mammary gland. A similar disorder to the lethal milk mouse occurs in humans. In the few cases studied, no changes in ZnT4 orthologue, hZnT4, were detected. This, and the presence of several minor phenotypic differences between the zinc deficiency in humans and mice, suggests that the human condition is caused by defects in genes that are yet to be identified. Taking into account the fact that there are no definitive tests for zinc deficiency and that this disorder can go undiagnosed, plus the recent identification of multiple members of the SCL30 and SLC39, it is likely that mutations in other genes may underlie additional inherited disorders of zinc deficiency.

Induction of epithelial to mesenchymal transition in PMC42-LA human breast carcinoma cells by carcinoma-associated fibroblast secreted factors

BackgroundBreast carcinoma is accompanied by changes in the acellular and cellular components of the microenvironment, the latter typified by a switch from fibroblasts to myofibroblasts.MethodsWe utilised conditioned media cultures, Western blot analysis and immunocytochemistry to investigate the differential effects of normal mammary fibroblasts (NMFs) and mammary cancer-associated fibroblasts (CAFs) on the phenotype and behaviour of PMC42-LA breast cancer cells. NMFs were obtained from a mammary gland at reduction mammoplasty, and CAFs from a mammary carcinoma after resection.ResultsWe found greater expression of myofibroblastic markers in CAFs than in NMFs. Medium from both CAFs and NMFs induced novel expression of α-smooth muscle actin and cytokeratin-14 in PMC42-LA organoids. However, although conditioned media from NMFs resulted in distribution of vimentin-positive cells to the periphery of PMC42-LA organoids, this was not seen with CAF-conditioned medium. Upregulation of vimentin was accompanied by a mis-localization of E-cadherin, suggesting a loss of adhesive function. This was confirmed by visualizing the change in active β-catenin, localized to the cell junctions in control cells/cells in NMF-conditioned medium, to inactive β-catenin, localized to nuclei and cytoplasm in cells in CAF-conditioned medium.ConclusionWe found no significant difference between the influences of NMFs and CAFs on PMC42-LA cell proliferation, viability, or apoptosis; significantly, we demonstrated a role for CAFs, but not for NMFs, in increasing the migratory ability of PMC42-LA cells. By concentrating NMF-conditioned media, we demonstrated the presence of factor(s) that induce epithelial-mesenchymal transition in NMF-conditioned media that are present at higher levels in CAF-conditioned media. Our in vitro results are consistent with observations in vivo showing that alterations in stroma influence the phenotype and behaviour of surrounding cells and provide evidence for a role for CAFs in stimulating cancer progression via an epithelial-mesenchymal transition. These findings have implications for our understanding of the roles of signalling between epithelial and stromal cells in the development and progression of mammary carcinoma.

Strategies for enhancing the phytoremediation of cadmium-contaminated agricultural soils by Solanum nigrum L

Field trials contribute practical information towards the development of phytoremediation strategies that cannot be provided by laboratory tests. We conducted field experiments utilizing the Cd hyperaccumulator plant Solanum nigrum L., on farmland contaminated with 1.91 mg kg(-1) Cd in the soil. Our study showed that S. nigrum has a relatively high biomass. Planting density had a significant effect on the plant biomass and thus on overall Cd accumulation. For double harvesting, an optimal cutting position influenced the amount of Cd extracted from soils. Double cropping was found to significantly increase the amount of Cd extracted by S. nigrum. Fertilizing had no significant effect on plant biomass or on the Cd remediation of the soil over the short-term period. Our study indicates that S. nigrum can accumulate Cd from soils where the concentrations are relatively low, and thus has application for use in decontamination of slightly to moderately Cd-contaminated soil.

Hormonal regulation of the Menkes and Wilson copper-transporting ATPases in human placental Jeg-3 cells

Copper deficiency during pregnancy results in early embryonic death and foetal structural abnormalities including skeletal, pulmonary and cardiovascular defects. During pregnancy, copper is transported from the maternal circulation to the foetus by mechanisms which have not been clearly elucidated. Two copper-transporting ATPases, Menkes (ATP7A; MNK) and Wilson (ATP7B; WND), are expressed in the placenta and both are involved in placental copper transport, as copper accumulates in the placenta in both Menkes and Wilson disease. The regulatory mechanisms of MNK and WND and their exact role in the placenta are unknown. Using a differentiated polarized Jeg-3 cell culture model of placental trophoblasts, MNK and WND were shown to be expressed within these cells. Distinct roles for MNK and WND are suggested on the basis of their opposing responses to insulin. Insulin and oestrogen increased both MNK mRNA and protein levels, altered the localization of MNK towards the basolateral membrane in a copper-independent manner, and increased the transport of copper across this membrane. In contrast, levels of WND were decreased in response to insulin, and the protein was located in a tight perinuclear region, with a corresponding decrease in copper efflux across the apical membrane. These results are consistent with a model of copper transport in the placenta in which MNK delivers copper to the foetus and WND returns excess copper to the maternal circulation. Insulin and oestrogen stimulate copper transport to the foetus by increasing the expression of MNK and reducing the expression of WND. These data show for the first time that MNK and WND are differentially regulated by the hormones insulin and oestrogen in human placental cells.

Defining the E-Cadherin Repressor Interactome in Epithelial-Mesenchymal Transition: The PMC42 Model as a Case Study

Epithelial-mesenchymal transition (EMT) is a feature of migratory cellular processes in all stages of life, including embryonic development and wound healing. Importantly, EMT features cluster with disease states such as chronic fibrosis and cancer. The dissolution of the E-cadherin-mediated adherens junction (AJ) is a key preliminary step in EMT and may occur early or late in the growing epithelial tumour. This is a first step for tumour cells towards stromal invasion, intravasation, extravasation and distant metastasis. The AJ may be inactivated in EMT by directed E-cadherin cleavage; however, it is increasingly evident that the majority of AJ changes are transcriptional and mediated by an expanding group of transcription factors acting directly or indirectly to repress E-cadherin expression. A review of the current literature has revealed that these factors may regulate each other in a hierarchical pattern where Snail1 (formerly Snail) and Snail2 (formerly Slug) are initially induced, leading to the activation of Zeb family members, TCF3, TCF4, Twist, Goosecoid and FOXC2. Within this general pathway, many inter-regulatory relationships have been defined which may be important in maintaining the EMT phenotype. This may be important given the short half-life of Snail1 protein. We have investigated these inter-regulatory relationships in the mesenchymal breast carcinoma cell line PMC42 (also known as PMC42ET) and its epithelial derivative, PMC42LA. This review also discusses several newly described regulators of E-cadherin repressors including oestrogen receptor-α and new discoveries in hypoxia- and growth factor-induced EMT. Finally, we evaluated how these findings may influence approaches to current cancer treatment.

Mammalian copper-transporting P-Type ATPases, ATP7A and ATP7B: Emerging roles

Copper (Cu) has a role in a diverse and increasing number of pathways, physiological and disease processes. These roles are testament to the fundamental importance of Cu in biology and the need to understand the mechanisms that regulate Cu homeostasis. The mammalian Cu-transporting P-type ATPases ATP7A and ATP7B are two key proteins that regulate the Cu status of the body. They transport Cu across cellular membranes for biosynthetic and protective functions, enabling Cu to fulfill its role as a catalytic and structural cofactor for many essential enzymes, and to prevent a toxic build-up of Cu inside cells. A variety of regulatory mechanisms operate at transcriptional and post-translational levels to ensure adequate Cu supplies for both physiological and pathophysiological processes. This review summarizes the recent literature that is revealing the emerging roles of the Cu-ATPases in health and disease.

Tissue Nonspecific Alkaline Phosphatase Is Activated via a Two-step Mechanism by Zinc Transport Complexes in the Early Secretory Pathway

A number of enzymes become functional by binding to zinc during their journey through the early secretory pathway. The zinc transporters (ZnTs) located there play important roles in this step. We have previously shown that two zinc transport complexes, ZnT5/ZnT6 heterodimers and ZnT7 homo-oligomers, are required for the activation of alkaline phosphatases, by converting them from the apo- to the holo-form. Here, we investigated the molecular mechanisms of this activation. ZnT1 and ZnT4 expressed in chicken DT40 cells did not contribute to the activation of tissue nonspecific alkaline phosphatase (TNAP). The reduced activity of TNAP in DT40 cells deficient in both ZnT complexes was not restored by zinc supplementation nor by exogenous expression of other ZnTs that increase the zinc content in the secretory pathway. Moreover, we showed that expression of ZnT5/ZnT6 heterodimers reconstituted with zinc transport-incompetent ZnT5 mutant failed to restore TNAP activity but could stabilize the TNAP protein as the apo-form, regardless of zinc status. These findings demonstrate that TNAP is activated not simply by passive zinc binding but by an elaborate two-step mechanism via protein stabilization followed by enzyme conversion from the apo- to the holo-form with zinc loaded by ZnT complexes in the early secretory pathway.