Mayuko Kumasaka

Beta-catenin inhibits melanocyte migration but induces melanoma metastasis.

The canonical Wnt signalling pathway induces the β-catenin/lymphoid enhancer factor transcription factors. It is activated in various cancers, most characteristically carcinomas, in which it promotes metastatic spread by increasing migration and/or invasion. The Wnt/β-catenin signalling pathway is frequently activated in melanoma, but the presence of β-catenin in the nucleus does not seem to be a sign of aggressiveness in these tumours. We found that, unlike its positive role in stimulating migration and invasion of carcinoma cells, β-catenin signalling decreased the migration of melanocytes and melanoma cell lines. In vivo, β-catenin signalling in melanoblasts reduced the migration of these cells, causing a white belly-spot phenotype. The inhibition by β-catenin of migration was dependent on MITF-M, a key transcription factor of the melanocyte lineage, and CSK, an Src-inhibitor. Despite reducing migration, β-catenin signalling promoted lung metastasis in the NRAS-driven melanoma murine model. Thus, β-catenin may have conflicting roles in the metastatic spread of melanoma, repressing migration while promoting metastasis. These results highlight that metastasis formation requires a series of successful cellular processes, any one of which may not be optimally efficient.

Automated Cell Tracking and Analysis in Phase-Contrast Videos (iTrack4U): Development of Java Software Based on Combined Mean-Shift Processes

Cell migration is a key biological process with a role in both physiological and pathological conditions. Locomotion of cells during embryonic development is essential for their correct positioning in the organism; immune cells have to migrate and circulate in response to injury. Failure of cells to migrate or an inappropriate acquisition of migratory capacities can result in severe defects such as altered pigmentation, skull and limb abnormalities during development, and defective wound repair, immunosuppression or tumor dissemination. The ability to accurately analyze and quantify cell migration is important for our understanding of development, homeostasis and disease. In vitro cell tracking experiments, using primary or established cell cultures, are often used to study migration as cells can quickly and easily be genetically or chemically manipulated. Images of the cells are acquired at regular time intervals over several hours using microscopes equipped with CCD camera. The locations (x,y,t) of each cell on the recorded sequence of frames then need to be tracked. Manual computer-assisted tracking is the traditional method for analyzing the migratory behavior of cells. However, this processing is extremely tedious and time-consuming. Most existing tracking algorithms require experience in programming languages that are unfamiliar to most biologists. We therefore developed an automated cell tracking program, written in Java, which uses a mean-shift algorithm and ImageJ as a library. iTrack4U is a user-friendly software. Compared to manual tracking, it saves considerable amount of time to generate and analyze the variables characterizing cell migration, since they are automatically computed with iTrack4U. Another major interest of iTrack4U is the standardization and the lack of inter-experimenter differences. Finally, iTrack4U is adapted for phase contrast and fluorescent cells.

Non-equilibrium atmospheric pressure plasmas modulate cell cycle-related gene expressions in melanocytic tumors of RET-transgenic mice

The incidence of cutaneous malignant melanoma is increasing at a greater rate than that of any other cancer in the world. However, an effective therapy for malignant melanoma has not been established. Recently, some studies have shown an antitumor effect of non‐equilibrium atmospheric pressure plasmas (NEAPPs) in vitro. Here, we examined the in vivo effect of NEAPP on cell cycle regulators, key elements for malignant transformation, in spontaneously developed benign melanocytic tumors in a hairless animal model. NEAPP irradiation decreased expression levels of cell cycle promoters, Cyclin D1, E1 and E2, and increased expression level of a cell cycle repressor, p27KIP1. Cyclin D1, E1 and E2 and p27KIP expression levels were associated with malignant transformation of the benign tumor in the animal model. Our results suggest that NEAPP irradiation suppresses malignant transformation of a benign melanocytic tumor via control of the expression levels of cell cycle regulators.

Comparison of Barium and Arsenic Concentrations in Well Drinking Water and in Human Body Samples and a Novel Remediation System for These Elements in Well Drinking Water.

Health risk for well drinking water is a worldwide problem. Our recent studies showed increased toxicity by exposure to barium alone (≤700 µg/L) and coexposure to barium (137 µg/L) and arsenic (225 µg/L). The present edition of WHO health-based guidelines for drinking water revised in 2011 has maintained the values of arsenic (10 µg/L) and barium (700 µg/L), but not elements such as manganese, iron and zinc. Nevertheless, there have been very few studies on barium in drinking water and human samples. This study showed significant correlations between levels of arsenic and barium, but not its homologous elements (magnesium, calcium and strontium), in urine, toenail and hair samples obtained from residents of Jessore, Bangladesh. Significant correlation between levels of arsenic and barium in well drinking water and levels in human urine, toenail and hair samples were also observed. Based on these results, a high-performance and low-cost adsorbent composed of a hydrotalcite-like compound for barium and arsenic was developed. The adsorbent reduced levels of barium and arsenic from well water in Bangladesh and Vietnam to <7 µg/L within 1 min. Thus, we have showed levels of arsenic and barium in humans and propose a novel remediation system.

Isolation and developmental expression of Mitf in Xenopus laevis

Mitf (gene for microphthalmia‐associated transcription factor) encodes a transcription factor of the basic/helix‐loop‐helix/leucine‐zipper family and is a key regulator during the development of two different types of melanin‐producing cell lineages, namely neural crest‐derived melanocytes/melanophores, and the retinal pigment epithelium (RPE) differentiated from the outer layer of the eye cup. Mitf‐deficient mice show a lack of melanocytes and small eyes caused by abnormal RPE development. An interesting feature of Mitf is the existence of multiple isoforms with different amino termini and their functions in the development of these melanin‐producing pigment cells. In this study, we isolated two Mitf homologues (XlMitfα and XlMitfβ) and their isoforms from Xenopus laevis. Alignment analysis of the amino acid sequences of the N‐termini suggests that these isoforms are homologues of mouse Mitf‐M (expressed specifically in the melanocyte lineage) and Mitf‐A (strongly expressed in the RPE, although this expression is ubiquitous). In Xenopus, XlMitfα is strongly expressed in the melanophore lineage (especially in premigratory melanoblasts) and the presumptive RPE and the epiphysis, in which melanin‐producing cells differentiate in some vertebrates. Conservation of the Mitf isoforms expected to possess specific functions in the development of melanin‐producing cells and of the expressions in such cell types in Xenopus suggest that XlMitf plays a central role in the development of melanin‐producing cell lineages, and that, as in mice and humans, most of the signaling molecules or transcription factors implicated genetically in the development of melanin‐producing cell lineages affect either Mitf expression or its function (Goding [2000] Genes Dev. 14:1712–1728). Developmental Dynamics 230:107–113, 2004.

Regulation of melanoblast and retinal pigment epithelium development by Xenopus laevis Mitf

Mitf is a central regulator of pigment cell development that is essential for the normal development of the melanocyte and retinal pigment epithelium (RPE) lineages. To understand better the role of Mitf, we have used the Xenopus laevis experimental system to allow a rapid examination of the role of Mitf in vivo. Here, we report the function of XlMitfα‐M on melanophore development and melanization compared with that of Slug that is expressed in neural crest cells. Overexpression of XlMitfα‐M led to an increase in melanophores that was partly contributed by an increase in Slug‐positive cells, indicating that XlMitfα‐M is a key regulator of melanocyte/melanophore development and melanization. Moreover, overexpression of a dominant‐negative form of XlMitfα led to a decrease in the number of melanophores and induced abnormal melanoblast migration. We also observed an induction of ectopic RPE and extended RPE by overexpression of XlMitfα‐M and possible interactions between XlMitfα and several eye‐related genes essential for normal eye development. Developmental Dynamics 234:523–534, 2005.

Bypassing melanocyte senescence by β-catenin: A novel way to promote melanoma

The Wnt/beta-catenin signaling pathway plays a key role in several cellular functions during embryonic development and adult homeostasis. The deregulation of this pathway may lead to the development of cancer, including melanoma. Deregulation of the Wnt/beta-catenin pathway occurs through either the induction/repression of, or specific mutations in, various members of this signaling pathway; this results in the stabilization of beta-catenin and its translocation from the cytoplasm to the nucleus, where it regulates transcription. Although nuclear beta-catenin is clearly involved in malignant transformation, the mechanism by which it exerts its effects remains elusive. This review focuses on the molecular and cellular mechanisms that are driven by beta-catenin and lead to melanocyte transformation. In particular, we describe how beta-catenin induces melanocyte immortalization, a novel activity of this multifunction protein. Finally, we discuss how beta-catenin-induced immortalization can cooperate with MAPKinase pathways to produce melanoma.

Arsenite-Mediated Promotion of Anchorage-Independent Growth of HaCaT Cells through Placental Growth Factor

Various cancers including skin cancer are increasing in 45 million people exposed to arsenic above the World Health Organizations guideline value of 10u2009μgu2009l(-1). However, there is limited information on key molecules regulating arsenic-mediated carcinogenesis. Our fieldwork in Bangladesh demonstrated that levels of placental growth factor (PlGF) in urine samples from residents of cancer-prone areas with arsenic-polluted drinking water were higher than those in urine samples from residents of an area that was not polluted with arsenic. Our experimental study in human nontumorigenic HaCaT skin keratinocytes showed that arsenite promoted anchorage-independent growth with increased expression and secretion of PlGF, a ligand of vascular endothelial growth factor receptor1 (VEGFR1), and increased VEGFR1/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) activities. The arsenite-mediated promotion of anchorage-independent growth was strongly inhibited by PlGF depletion with decreased activities of the PlGF/VEGFR1/MEK/ERK pathway. Moreover, arsenite proteasome-dependently degrades metal-regulatory transcription factor-1 (MTF-1) protein, resulting in a decreased amount of MTF-1 protein binding to the PlGF promoter. MTF-1 negatively controlled PlGF transcription in HaCaT cells, resulting in increased PlGF transcription. These results suggest that arsenite-mediated MTF-1 degradation enhances the activity of PlGF/VEGFR1/MEK/ERK signaling, resulting in promotion of the malignant transformation of keratinocytes. Thus, this study proposed a molecular mechanism for arsenite-mediated development of skin cancer.

Bidirectional functions of arsenic as a carcinogen and an anti-cancer agent in human squamous cell carcinoma.

Bidirectional cancer-promoting and anti-cancer effects of arsenic for cancer cells have been revealed in previous studies. However, each of these effects (cancer-promoting or anti-cancer) was found in different cells at different treated-concentration of arsenic. In this study, we for the first time indicated that arsenic at concentration of 3 µM, equal to average concentration in drinking water in cancer-prone areas in Bangladesh, simultaneously expressed its bidirectional effects on human squamous cell carcinoma HSC5 cells with distinct pathways. Treatment with 3 µM of arsenic promoted cell invasion via upregulation of expression of MT1-MMP and downregulation of expression of p14ARF and simultaneously induced cell apoptosis through inhibition of expression of N-cadherin and increase of expression of p21(WAF1/CIP1) at both transcript and protein levels in HSC5 cells. We also showed that inhibition of MT1-MMP expression by NSC405020 resulted in decrease of arsenic-mediated invasion of HSC5 cells involving decrease in phosphorylated extracellular signal-regulated kinases (pERK). Taken together, our biological and biochemical findings suggested that arsenic expressed bidirectional effects as a carcinogen and an anti-cancer agent in human squamous cell carcinoma HSC5 cells with distinct pathways. Our results might play an important scientific evident for further studies to find out a better way in treatment of arsenic-induced cancers, especially in squamous cell carcinoma.

Commentary to Krishna et al. (2014): Brain deposition and neurotoxicity of manganese in adult mice exposed via the drinking water

Krishna et al. (Arch Toxicol 88(1):47–64, 2014) recently published the results of a study in which adult C57BL/6 mice were subchronically exposed to 400,000xa0μg/L manganese (Mn) using manganese chloride via drinking water for 8xa0weeks and examined the neurotoxic effects. After 5xa0weeks of Mn exposure, significant deposition of Mn in all of the brain regions examined by magnetic resonance imaging was detected. After 6xa0weeks of Mn exposure, neurobehavioral deficits in an open field test, a grip strength test, and a forced swim test were observed. Eight weeks of Mn exposure increased striatal 5-hydroxyindoleacetic acid (a serotonin metabolite) levels, but did not alter the levels of striatal dopamine, its metabolites and serotonin. Krishna et al. also reported significant increases in mRNA levels of GFAP (an astrocyte activation marker), HO-1 (an oxidative stress marker) and NOS2 (a nitrosative stress marker), and in protein expression level of GFAP in the substantia nigra pars reticulata after 8xa0weeks of Mn exposure. These results suggest that 400,000xa0μg/L Mn exposure via drinking water in mice induces neurobehavioral deficits, serotonergic imbalance, and glial activation accompanied by an increase in brain Mn deposition. The report by Krishna et al. is interesting because the studies on the neurobehavioral effect of Mn exposure by drinking water in mice are very limited. However, Mn concentrations previously reported in well drinking water (Agusa et al. in Vietnam Environ Pollut 139(1):95–106, 2006; Buschmann et al. in Environ Int 34(6):756–764, 2008; Hafeman et al. in Environ Health Perspect 115(7):1107–1112, 2007; Wasserman et al. in Bangladesh Environ Health Perspect 114(1):124–129, 2006) were lower than 400,000xa0μg/L.