MiTF links Erk1/2 kinase and p21CIP1/WAF1 activation after UVC radiation in normal human melanocytes and melanoma cells

As a survival factor for melanocytes lineage cells, MiTF plays multiple roles in development and melanomagenesis. What role MiTF plays in the DNA damage response is currently unknown. In this report we observed that MiTF was phosphorylated at serine 73 after UVC radiation, which was followed by proteasome-mediated degradation. Unlike after c-Kit stimulation, inhibiting p90RSK-1 did not abolish the band shift of MiTF protein, nor did it abolish the UVC-mediated MiTF degradation, suggesting that phosphorylation on serine 73 by Erk1/2 is a key event after UVC. Furthermore, the MiTF-S73A mutant (Serine 73 changed to Alanine via site-directed mutagenesis) was unable to degrade and was continuously expressed after UVC exposure. Compared to A375 melanoma cells expressing wild-type MiTF (MiTF-WT), cells expressing MiTF-S73A mutant showed less p21WAF1/CIP1 accumulation and a delayed p21WAF1/CIP1 recovery after UVC. Consequently, cells expressing MiTF-WT showed a temporary G1 arrest after UVC, but cells expressing MiTF-S73A mutant or lack of MiTF expression did not. Finally, cell lines with high levels of MiTF expression showed higher resistance to UVC-induced cell death than those with low-level MiTF. These data suggest that MiTF mediates a survival signal linking Erk1/2 activation and p21WAF1/CIP1 regulation via phosphorylation on serine 73, which facilitates cell cycle arrest. In addition, our data also showed that exposure to different wavelengths of UV light elicited different signal pathways involving MiTF

Structural constraints revealed in consistent nucleosome positions in the genome of S. cerevisiae

<p>Abstract</p> <p>Background</p> <p>Recent advances in the field of high-throughput genomics have rendered possible the performance of genome-scale studies to define the nucleosomal landscapes of eukaryote genomes. Such analyses are aimed towards providing a better understanding of the process of nucleosome positioning, for which several models have been suggested. Nevertheless, questions regarding the sequence constraints of nucleosomal DNA and how they may have been shaped through evolution remain open. In this paper, we analyze in detail different experimental nucleosome datasets with the aim of providing a hypothesis for the emergence of nucleosome-forming sequences.</p> <p>Results</p> <p>We compared the complete sets of nucleosome positions for the budding yeast (<it>Saccharomyces cerevisiae</it>) as defined in the output of two independent experiments with the use of two different experimental techniques. We found that < 10% of the experimentally defined nucleosome positions were consistently positioned in both datasets. This subset of well-positioned nucleosomes, when compared with the bulk, was shown to have particular properties at both sequence and structural levels. Consistently positioned nucleosomes were also shown to occur preferentially in pairs of dinucleosomes, and to be surprisingly less conserved compared with their adjacent nucleosome-free linkers.</p> <p>Conclusion</p> <p>Our findings may be combined into a hypothesis for the emergence of a weak nucleosome-positioning code. According to this hypothesis, consistent nucleosomes may be partly guided by nearby nucleosome-free regions through statistical positioning. Once established, a set of well-positioned consistent nucleosomes may impose secondary constraints that further shape the structure of the underlying DNA. We were able to capture these constraints through the application of a recently introduced structural property that is related to the symmetry of DNA curvature. Furthermore, we found that both consistently positioned nucleosomes and their adjacent nucleosome-free regions show an increased tendency towards conservation of this structural feature.</p

β-Catenin Signaling Increases during Melanoma Progression and Promotes Tumor Cell Survival and Chemoresistance

Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy

Kita Driven Expression of Oncogenic HRAS Leads to Early Onset and Highly Penetrant Melanoma in Zebrafish

Melanoma is the most aggressive and lethal form of skin cancer. Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed.Using the combinatorial Gal4-UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth. By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish. The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors. In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period.This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors. Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens

Inhibition of Melanogenesis by the Pyridinyl Imidazole Class of Compounds: Possible Involvement of the Wnt/β-Catenin Signaling Pathway

While investigating the role of p38 MAPK in regulating melanogenesis, we found that pyridinyl imidazole inhibitors class compounds as well as the analog compound SB202474, which does not inhibit p38 MAPK, suppressed both α-MSH-induced melanogenesis and spontaneous melanin synthesis. In this study, we demonstrated that the inhibitory activity of the pyridinyl imidazoles correlates with inhibition of the canonical Wnt/β-catenin pathway activity. Imidazole-treated cells showed a reduction in the level of Tcf/Lef target genes involved in the β-catenin signaling network, including ubiquitous genes such as Axin2, Lef1, and Wisp1 as well as cell lineage-restricted genes such as microphthalmia-associated transcription factor and dopachrome tautomerase. Although over-expression of the Wnt signaling pathway effector β-catenin slightly restored the melanogenic program, the lack of complete reversion suggested that the imidazoles interfered with β-catenin-dependent transcriptional activity rather than with β-catenin expression. Accordingly, we did not observe any significant change in β-catenin protein expression. The independence of p38 MAPK activity from the repression of Wnt/β-catenin signaling pathway was confirmed by small interfering RNA knockdown of p38 MAPK expression, which by contrast, stimulated β-catenin-driven gene expression. Our data demonstrate that the small molecule pyridinyl imidazoles possess two distinct and opposite mechanisms that modulate β-catenin dependent transcription: a p38 inhibition-dependent effect that stimulates the Wnt pathway by increasing β-catenin protein expression and an off-target mechanism that inhibits the pathway by repressing β-catenin protein functionality. The p38-independent effect seems to be dominant and, at least in B16-F0 cells, results in a strong block of the Wnt/β-catenin signaling pathway

Predicting Human Nucleosome Occupancy from Primary Sequence

Nucleosomes are the fundamental repeating unit of chromatin and comprise the structural building blocks of the living eukaryotic genome. Micrococcal nuclease (MNase) has long been used to delineate nucleosomal organization. Microarray-based nucleosome mapping experiments in yeast chromatin have revealed regularly-spaced translational phasing of nucleosomes. These data have been used to train computational models of sequence-directed nuclesosome positioning, which have identified ubiquitous strong intrinsic nucleosome positioning signals. Here, we successfully apply this approach to nucleosome positioning experiments from human chromatin. The predictions made by the human-trained and yeast-trained models are strongly correlated, suggesting a shared mechanism for sequence-based determination of nucleosome occupancy. In addition, we observed striking complementarity between classifiers trained on experimental data from weakly versus heavily digested MNase samples. In the former case, the resulting model accurately identifies nucleosome-forming sequences; in the latter, the classifier excels at identifying nucleosome-free regions. Using this model we are able to identify several characteristics of nucleosome-forming and nucleosome-disfavoring sequences. First, by combining results from each classifier applied de novo across the human ENCODE regions, the classifier reveals distinct sequence composition and periodicity features of nucleosome-forming and nucleosome-disfavoring sequences. Short runs of dinucleotide repeat appear as a hallmark of nucleosome-disfavoring sequences, while nucleosome-forming sequences contain short periodic runs of GC base pairs. Second, we show that nucleosome phasing is most frequently predicted flanking nucleosome-free regions. The results suggest that the major mechanism of nucleosome positioning in vivo is boundary-event-driven and affirm the classical statistical positioning theory of nucleosome organization

Integrative Analysis of Epigenetic Modulation in Melanoma Cell Response to Decitabine: Clinical Implications

Decitabine, an epigenetic modifier that reactivates genes otherwise suppressed by DNA promoter methylation, is effective for some, but not all cancer patients, especially those with solid tumors. It is commonly recognized that to overcome resistance and improve outcome, treatment should be guided by tumor biology, which includes genotype, epigenotype, and gene expression profile. We therefore took an integrative approach to better understand melanoma cell response to clinically relevant dose of decitabine and identify complementary targets for combined therapy. We employed eight different melanoma cell strains, determined their growth, apoptotic and DNA damage responses to increasing doses of decitabine, and chose a low, clinically relevant drug dose to perform whole-genome differential gene expression, bioinformatic analysis, and protein validation studies. The data ruled out the DNA damage response, demonstrated the involvement of p21Cip1 in a p53-independent manner, identified the TGFβ pathway genes CLU and TGFBI as markers of sensitivity to decitabine and revealed an effect on histone modification as part of decitabine-induced gene expression. Mutation analysis and knockdown by siRNA implicated activated β-catenin/MITF, but not BRAF, NRAS or PTEN mutations as a source for resistance. The importance of protein stability predicted from the results was validated by the synergistic effect of Bortezomib, a proteasome inhibitor, in enhancing the growth arrest of decitabine in otherwise resistant melanoma cells. Our integrative analysis show that improved therapy can be achieved by comprehensive analysis of cancer cells, identified biomarkers for patient's selection and monitoring response, as well as targets for improved combination therapy

Legal and ethical framework for global health information and biospecimen exchange - an international perspective.

BACKGROUND: The progress of electronic health technologies and biobanks holds enormous promise for efficient research. Evidence shows that studies based on sharing and secondary use of data/samples have the potential to significantly advance medical knowledge. However, sharing of such resources for international collaboration is hampered by the lack of clarity about ethical and legal requirements for transfer of data and samples across international borders. MAIN TEXT: Here, the International Clinical Trial Center Network (ICN) reports the legal and ethical requirements governing data and sample exchange (DSE) across four continents. The most recurring requirement is ethical approval, whereas only in specific conditions approval of national health authorities is required. Informed consent is not required in all sharing situations. However, waiver of informed consent is only allowed in certain countries/regions and under certain circumstances. The current legal and ethical landscape appears to be very complex and under constant evolution. Regulations differ between countries/regions and are often incomplete, leading to uncertainty. CONCLUSION: With this work, ICN illuminates the unmet need for a single international collaborative framework to facilitate DSE. Harmonising requirements for global DSE will reduce inefficiency and waste in research. There are many challenges to realising this ambitious vision, including inconsistent terminology and definitions, and heterogeneous and dynamic legal constraints. Here, we identify areas of agreement and significant difference as a necessary first step towards facilitating international collaboration. We propose the establishment of a working group to continue the comparison across jurisdictions, create a standardised glossary and define a set of basic principles and fundamental requirements for DSE

Skipping of Exons by Premature Termination of Transcription and Alternative Splicing within Intron-5 of the Sheep SCF Gene: A Novel Splice Variant

Stem cell factor (SCF) is a growth factor, essential for haemopoiesis, mast cell development and melanogenesis. In the hematopoietic microenvironment (HM), SCF is produced either as a membrane-bound (−) or soluble (+) forms. Skin expression of SCF stimulates melanocyte migration, proliferation, differentiation, and survival. We report for the first time, a novel mRNA splice variant of SCF from the skin of white merino sheep via cloning and sequencing. Reverse transcriptase (RT)-PCR and molecular prediction revealed two different cDNA products of SCF. Full-length cDNA libraries were enriched by the method of rapid amplification of cDNA ends (RACE-PCR). Nucleotide sequencing and molecular prediction revealed that the primary 1519 base pair (bp) cDNA encodes a precursor protein of 274 amino acids (aa), commonly known as ‘soluble’ isoform. In contrast, the shorter (835 and/or 725 bp) cDNA was found to be a ‘novel’ mRNA splice variant. It contains an open reading frame (ORF) corresponding to a truncated protein of 181 aa (vs 245 aa) with an unique C-terminus lacking the primary proteolytic segment (28 aa) right after the D175G site which is necessary to produce ‘soluble’ form of SCF. This alternative splice (AS) variant was explained by the complete nucleotide sequencing of splice junction covering exon 5-intron (5)-exon 6 (948 bp) with a premature termination codon (PTC) whereby exons 6 to 9/10 are skipped (Cassette Exon, CE 6–9/10). We also demonstrated that the Northern blot analysis at transcript level is mediated via an intron-5 splicing event. Our data refine the structure of SCF gene; clarify the presence (+) and/or absence (−) of primary proteolytic-cleavage site specific SCF splice variants. This work provides a basis for understanding the functional role and regulation of SCF in hair follicle melanogenesis in sheep beyond what was known in mice, humans and other mammals