HJURP is recruited to double-strand break sites and facilitates DNA repair by promoting chromatin reorganization.

HJURP is overexpressed in several cancer types and strongly correlates with patient survival. However, the mechanistic basis underlying the association of HJURP with cancer aggressiveness is not well understood. HJURP promotes the loading of the histone H3 variant, CENP-A, at the centromeric chromatin, epigenetically defining the centromeres and supporting proper chromosome segregation. In addition, HJURP is associated with DNA repair but its function in this process is still scarcely explored. Here, we demonstrate that HJURP is recruited to DSBs through a mechanism requiring chromatin PARylation and promotes epigenetic alterations that favor the execution of DNA repair. Incorporation of HJURP at DSBs promotes turnover of H3K9me3 and HP1, facilitating DNA damage signaling and DSB repair. Moreover, HJURP overexpression in glioma cell lines also affected global structure of heterochromatin independently of DNA damage induction, promoting genome-wide reorganization and assisting DNA damage response. HJURP overexpression therefore extensively alters DNA damage signaling and DSB repair, and also increases radioresistance of glioma cells. Importantly, HJURP expression levels in tumors are also associated with poor response of patients to radiation. Thus, our results enlarge the understanding of HJURP involvement in DNA repair and highlight it as a promising target for the development of adjuvant therapies that sensitize tumor cells to irradiation.

HJURP knockdown disrupts clonogenic capacity and increases radiation-induced cell death of glioblastoma cells.

The Holliday Junction-Recognition Protein (HJURP) was reported as overexpressed in several cancers and also strongly correlated with poor prognosis of patients, especially in glioblastoma (GBM), the most common and deadly type of primary brain tumor. HJURP is responsible for loading the histone H3 variant-the Centromeric Protein A (CENP-A)-at the centromeres in a cell cycle-regulated manner, being required for proper chromosome segregation. Here we investigated HJURP association with survival and radioresistance of different GBM cell lines. HJURP knockdown compromised the clonogenic capacity and severely impaired survival of five distinct GBM cells, while nontumor astrocytes were not affected. U251MG cells showed a robust cell cycle arrest in G2/M phases followed by a drastic increment in cell death after HJURP silencing, while U138MG and U343MG cell lines presented augmented senescence with a comparable increase in cell death. Importantly, we verified that the impact on cell cycle dynamics and clonogenic survival were associated with loss CENP-A at the centromeres. Moreover, radiation resistance was also impacted by HJURP modulation in several GBM cell lines. U87MG, T98G, U138MG, and U343MG cells were all sensitized to ionizing radiation after HJURP reduction. These data reinforce the requirement of HJURP for proliferative capacity and radioresistance of tumor cells, underlining its potential as a promising therapeutic target for GBM.

Myosin Va interacts with the exosomal protein spermine synthase.

Myosin Va (MyoVa) is an actin-based molecular motor that plays key roles in the final stages of secretory pathways, including neurotransmitter release. Several studies have addressed how MyoVa coordinates the trafficking of secretory vesicles, but why this molecular motor is found in exosomes is still unclear. In this work, using a yeast two-hybrid screening system, we identified the direct interaction between the globular tail domain (GTD) of MyoVa and four protein components of exosomes: the WD repeat-containing protein 48 (WDR48), the cold shock domain-containing protein E1 (CSDE1), the tandem C2 domain-containing protein 1 (TC2N), and the enzyme spermine synthase (SMS). The interaction between the GTD of MyoVa and SMS was further validated in vitro and displayed a Kd in the low micromolar range (3.5 ± 0.5 µM). SMS localized together with MyoVa in cytoplasmic vesicles of breast cancer MCF-7 and neuroblastoma SH-SY5Y cell lines, known to produce exosomes. Moreover, MYO5A knockdown decreased the expression of SMS gene and rendered the distribution of SMS protein diffuse, supporting a role for MyoVa in SMS expression and targeting.

The lncRNA RMEL3 protects immortalized cells from serum withdrawal-induced growth arrest and promotes melanoma cell proliferation and tumor growth.

RMEL3 is a recently identified lncRNA associated with BRAFV600E mutation and melanoma cell survival. Here, we demonstrate strong and moderate RMEL3 upregulation in BRAF and NRAS mutant melanoma cells, respectively, compared to melanocytes. High expression is also more frequent in cutaneous than in acral/mucosal melanomas, and analysis of an ICGC melanoma dataset showed that mutations in RMEL3 locus are preponderantly C > T substitutions at dipyrimidine sites including CC > TT, typical of UV signature. RMEL3 mutation does not correlate with RMEL3 levels, but does with poor patient survival, in TCGA melanoma dataset. Accordingly, RMEL3 lncRNA levels were significantly reduced in BRAFV600E melanoma cells upon treatment with BRAF or MEK inhibitors, supporting the notion that BRAF-MEK-ERK pathway plays a role to activate RMEL3 gene transcription. RMEL3 overexpression, in immortalized fibroblasts and melanoma cells, increased proliferation and survival under serum starvation, clonogenic ability, and xenografted melanoma tumor growth. Although future studies will be needed to elucidate the mechanistic activities of RMEL3, our data demonstrate that its overexpression bypasses the need of mitogen activation to sustain proliferation/survival of non-transformed cells and suggest an oncogenic role for RMEL3.

RMEL3, a novel BRAFV600E-associated long noncoding RNA, is required for MAPK and PI3K signaling in melanoma.

Previous work identified RMEL3 as a lncRNA with enriched expression in melanoma. Analysis of The Cancer Genome Atlas (TCGA) data confirmed RMEL3 enriched expression in melanoma and demonstrated its association with the presence of BRAFV600E. RMEL3 siRNA-mediated silencing markedly reduced (95%) colony formation in different BRAFV600E melanoma cell lines. Multiple genes of the MAPK and PI3K pathways found to be correlated with RMEL3 in TCGA samples were experimentally confirmed. RMEL3 knockdown led to downregulation of activators or effectors of these pathways, including FGF2, FGF3, DUSP6, ITGB3 and GNG2. RMEL3 knockdown induces gain of protein levels of tumor suppressor PTEN and the G1/S cyclin-Cdk inhibitors p21 and p27, as well as a decrease of pAKT (T308), BRAF, pRB (S807, S811) and cyclin B1. Consistently, knockdown resulted in an accumulation of cells in G1 phase and subG0/G1 in an asynchronously growing population. Thus, TCGA data and functional experiments demonstrate that RMEL3 is required for MAPK and PI3K signaling, and its knockdown decrease BRAFV600E melanoma cell survival and proliferation.

Development, characterization, and photocytotoxicity assessment on human melanoma of chloroaluminum phthalocyanine nanocapsules.

In this work we have developed nanocapsules containing chloroaluminum phthalocyanine (ClAlPc) and assessed their phototoxic action on WM1552C, WM278, and WM1617 human melanoma cell lines. The ClAlPc-loaded nanocapsules were prepared by the nanoprecipitation method and optimized by means of a 2(3) full factorial design. The ClAlPc nanocapsules were characterized by particle size and distribution, zeta potential, morphology, encapsulation efficiency, singlet oxygen production, stability, and phototoxic action on melanoma cells. Both the development and optimization studies revealed that stable colloidal formulations could be obtained by using 1.75% (w/v) soybean lecithin, 1.25% (w/v) Poloxamer 188, 2.5% (v/v) soybean oil, and 0.75% (w/v) poly(D,L-lactide-co-glycolide). The nanocapsules had a mean diameter of 230 nm, homogeneous size distribution (polydispersity index<0.3), and negative zeta potential (about -30 mV). Their morphology was spherical, with evident polymer membrane coating droplet. The encapsulation efficiency was 70%, as expected for hydrophobic drugs, and the nanoencapsulated ClAlPc was able to produce high singlet oxygen quantum yield. ClAlPc nanocapsules exhibited good physical stability over a 12-month period. WM1552C primary melanoma cells were more sensitive (p<0.05) to the phototoxic effect elicited by ClAlPc nanocapsules (0.3 µg ml(-1)) under light irradiation at 20 mJ cm(-2). On the other hand, the cell survival percentage for all the melanoma cell lines treated with the highest light dose (150 mJ cm(-2)) was lower than 10%. In summary, ClAlPc nanoencapsulation could enable application of this hydrophobic photosensitizer in the treatment of malignant melanoma with the use of both low sensitizer drug concentration and light dose.

trans-[Ru(NO)Cl(cyclam)](PF6)2 and [Ru(NO)(Hedta)] incorporated in PLGA nanoparticles for the delivery of nitric oxide to B16-F10 cells: cytotoxicity and phototoxicity.

The immobilization and characterization of trans-[Ru(NO)Cl(cyclam)](PF6)2 (cyclam=1,4,8,11-tetraazacyclotetradecane), and [Ru(NO)(Hedta)] (Hedta=ethylenediaminetetraacetic acid) entrapped in poly(d,l-lactic-co-glycolic) acid (PLGA) nanoparticles (NP) using the double emulsification process is described. Scanning electron microscopy and dynamic light scattering revealed that the particles are spherical in shape, have a size distribution between 220 and 840 nm of diameter, and have a tendency to aggregate confirmed by a zeta potential between -3.2 and +3.5 mV. Using this method the loading efficiency was 26% for trans-[Ru(NO)Cl(cyclam)](PF6)2 and 32% for [Ru(NO)(Hedta)]. The release of the complexes from the NPs shows that cyclam-NP and Hedta-NP exhibited a two-phase exponential association release pattern, which was characterized by an initial complex burst during the first 24 h, followed by a slower release phase complex profile, due to a few pores observed in surface of nanoparticles using atomic force microscopy. The in vitro cytotoxic activity of the nitrosyl complexes in solution and incorporated in PLGA nanoparticles on melanoma cancer cells (cell line B16-F10) was investigated. The lower cytotoxicity of trans-[RuCl(cyclam)(NO)]2+ (12.4±2.6%) and [Ru(NO)(Hedta)] (4.0±2.7%) in solution compared to that of trans-[Ru(NO)(NH3)4py]3+ (46.1±6.4%) is consistent with the rate constant release of NO of these complexes (k-NO=6.2×10(-4) s(-1), 2.0×10(-3) s(-1), and 6.0×10(-2) s(-1), respectively); the cytotoxicities are also inhibited in the presence of the NO scavenger carboxy-PTIO. The phototoxicity of these complexes is due to NO release, which lead to 53.8±6.2% of cell death in the presence of trans-[Ru(NO)Cl(cyclam)](PF6)2 and 22.3±5.1% in the presence of [Ru(NO)(Hedta)]. The PLGA nanoparticles loaded with trans-[Ru(NO)Cl(cyclam)](PF6)2 and [Ru(NO)(Hedta)] exerted in vitro a reduced activity against melanoma cells when compared to the activity of complex in solution (nonentrapped in nanoparticles). Blank PLGA nanoparticles did not exhibit cytotoxicity. In the presence of light and of ruthenium nitrosyl complexes or cyclam-NP and Hedta-NP, B16-F10 cells displayed a considerable damage of the surface with rupture of the plasma membrane. This behavior is an indicative of the efficiency of the DDS to deliver the NO from the entrapped complex when photoinduced.

Novel primate-specific genes, RMEL 1, 2 and 3, with highly restricted expression in melanoma, assessed by new data mining tool.

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.

In vitro phototoxicity of liposomes and nanocapsules containing chloroaluminum phthalocyanine on human melanoma cell line.

In this study, the photodynamic action of liposomes (LP) and nanocapsules (NC) containing Chloroaluminum phthalocyanine (CIAIPc), on the human melanoma cell (WM 1552C), was assessed. The light source was setup at 672 nm, which corresponds to the maximum absorption wavelength of the CIAIPc. Both colloidal carriers presented size in nanometric scale as well as negative zeta potential. The cellular damage was light dose dependent ranging from 30% of cell death at 70 mJ x cm-2 to 90% of death at 700 mJ x cm(-2). However, the photocytotoxic effect of LP at 70 mJ x cm(-2) was slightly more efficient to induce cellular death than NC formulation. At 140 mJ x cm(-2), and 700 mJ x cm(-2) both nanocarriers were equally efficient to induce cellular damage. Therefore, in the present work, the maximum phototoxic effect was obtained with 700 mJ x cm(-2) of light dose, in combination with 0.29 microg x mL(-1) of CIAIPc encapsulated into LP and NC. The cells were also positive to annexin V, after the PDT treatment with LP and NC, showing that one of the mechanisms of cellular death involved is apoptosis. In summary, the potential of LP and NC as a drug delivery system, in Photodynamic Therapy (PDT) against melanoma, has been confirmed using a lower concentration of the photosensitizer and lower light doses than that applied in current protocols. This is an innovative proposal to treat melanoma cell lines that until now have not received the benefit of the PDT protocol for treatment.

Myosin Va phosphorylated on Ser1650 is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcription.

Nuclear actin and nuclear myosins have been implicated in the regulation of gene expression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser(1650) MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine(1650) and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser(1650) MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser(1650) MVa to nucleoli, as well as separating a fraction of phospho-ser(1650) MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation.

trans-[Ru(NO)(NH3)4(py)](BF4)3.H2O encapsulated in PLGA microparticles for delivery of nitric oxide to B16-F10 cells: Cytotoxicity and phototoxicity.

The NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (py=pyridine) was loaded into poly-lactic-co-glycolic acid (PLGA) microparticles using the double emulsification technique. Scanning electron microscopy (SEM) and dynamic light scattering revealed that the particles are spherical in shape, have a diameter of 1600nm, and have low tendency to aggregate. The entrapment efficiency was 25%. SEM analysis of the melanoma cell B16-F10 in the presence of the microparticles containing the complex trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (pyMP) showed that the microparticles were adhered to the cell surface after 2h of incubation. The complex with concentrations lower than 1x10(-4)M did not show toxicity in B16-F10 murine cells. The complex in solution is toxic at higher concentrations (>1x10(-3)M), with cell death attributed to NO release following the reduction of the complex. pyMP is not cytotoxic due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. However, pyMP is phototoxic upon light irradiation. The phototoxicity strongly suggests that cell death is due to NO release from trans-[Ru(NO)(NH(3))(4)(py)](3+). This work shows that pyMP can serve as a model for a drug delivery system carrying the NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O, which can release NO locally at the tumor cell by irradiation with light only.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets.

BACKGROUND: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alphavbeta3-integrin and low levels of RHOC. METHODS: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. RESULTS: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. CONCLUSION: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

A transcript finishing initiative for closing gaps in the human transcriptome.

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms.

Griscelli syndrome: characterization of a new mutation and rescue of T-cytotoxic activity by retroviral transfer of RAB27A gene.

Griscelli syndrome (GS) is caused by mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes, all of which lead to a similar pigmentary dilution. In addition, GS1 patients show primary neurological impairment, whereas GS2 patients present immunodeficiency and periods of lymphocyte proliferation and activation, leading to their infiltration in many organs, such as the nervous system, causing secondary neurological damage. We report the diagnosis of GS2 in a 4-year-old child with haemophagocytic syndrome, immunodeficiency, and secondary neurological disorders. Typical melanosome accumulation was found in skin melanocytes and pigment clumps were observed in hair shafts. Two heterozygous mutant alleles of the RAB27A gene were found, a C-T transition (C352T) that leads to Q118stop and a G-C transversion on the exon 5 splicing donor site (G467+1C). Functional assays showed increased cellular activation and decreased cytotoxic activity of NK and CD8+ T cells, associated with defective lytic granules release. Myosin-Va expression and localization in the patient lymphocytes were also analyzed. Most importantly, we show that cytotoxic activity of the patient's CD8+ T lymphocytes can be rescued in vitro by RAB27A gene transfer mediated by a recombinant retroviral vector, a first step towards a potential treatment of the acute phase of GS2 by RAB27A transduced lymphocytes.

A experiência do estudante de graduaçäo de enfermagem na Escola de 1§ grau prestando assistência primária de saúde / Experience of an undergraduate student of nursing in a school of 1st degree giving primary health care

O trabalho analisa a experiência de 43 dos 65 estudantes que cursam o 5§ semestre de graduaçäo de 1982 da Escola de Enfermagem da USP, Campus Ribeiräo Preto, atuando em uma escola de 1§ grau, localizada na periferia de Ribeiräo Preto, constituída por alunos do bairro de nível sócio-econômico precário prestando assistência de saúde ao escolar, através de seu meio-ambiente: escola, família e comunidade. Para avaliar a experiência desses estudantes, utilizamos um questionário constando de 18 questöes. A análise dos resultados traduz a importância de se incluir programas semelhantes nos currículos de graduaçäo de enfermagem, significando aprendizado, conscientizaçäo dos estudantes e abertura de campo de atuaçäo do enfermeiro