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In the original publication [...].
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The highly dynamic nature of chromatin's structure, due to the epigenetic alterations of histones and DNA, controls cellular plasticity and allows the rewiring of the epigenetic landscape required for either cell differentiation or cell (re)programming. To dissect the epigenetic switch enabling the programming of a cancer cell, we carried out wide genome analysis of Histone 3 (H3) modifications during osteogenic differentiation of SH-SY5Y neuroblastoma cells. The most significant modifications concerned H3K27me2/3, H3K9me2, H3K79me1/2, and H3K4me1 that specify the process of healthy adult stem cell differentiation. Next, we translated these findings in vivo, assessing H3K27, H3K9, and H3K79 methylation states in biopsies derived from patients affected by basalioma, head and neck carcinoma, and bladder tumors. Interestingly, we found a drastic decrease in H3K9me2 and H3K79me3 in cancer specimens with respect to their healthy counterparts and also a positive correlation between these two epigenetic flags in all three tumors. Therefore, we suggest that elevated global levels of H3K9me2 and H3K79me3, present in normal differentiated cells but lost in malignancy, may reflect an important epigenetic barrier to tumorigenesis. This suggestion is further corroborated, at least in part, by the deranged expression of the most relevant H3 modifier enzymes, as revealed by bioinformatic analysis. Overall, our study indicates that the simultaneous occurrence of H3K9me2 and H3K79me3 is fundamental to ensure the integrity of differentiated tissues and, thus, their combined evaluation may represent a novel diagnostic marker and potential therapeutic target.
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Neuroblastoma , Osteogênese , Adulto , Humanos , Neuroblastoma/genética , Histonas/metabolismo , Transformação Celular Neoplásica/genética , Epigênese GenéticaRESUMO
Ichthyoses are genetically determined cornification disorders of the epidermis characterized by the presence of different degrees of scaling, hyperkeratosis, and erythroderma often associated with palmoplantar keratoderma. Different classifications of these diseases have been proposed, often based upon the involved genes and/or the clinical presentation. The clinical features of these diseases present some overlap of phenotypes among distinct genetic entities, depending mainly on the penetrance of mutations. In this study, using a clinical, genetic, and molecular approach, we analyzed a family with two affected members who had clinical and histological features resembling erythrokeratodermia variabilis (EKV) or a type of erythrodermic hyperkeratosis with palmoplantar keratoderma. Despite of the clinical presentation, we demonstrated that the affected patients were genetically double heterozygous for two different mutations in the ABCA12 gene, known to be responsible for harlequin ichthyosis. To explain the mild phenotype of our patients, we performed a molecular characterization of the skin. In the upper layers of the epidermis, the results showed a patchy presence of the glucosyl-ceramides (GlcCer), which is the lipid transported by ABCA12, fundamental in contributing to skin impermeability. Indeed, the two mutations detected do not completely abolish ABCA12 activity, indicating that the mild phenotype is due to a partial loss of function of the enzyme, thus giving rise to an intermediate phenotype resembling EKVP, due to a partial depletion of GlcCer deposition.
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Eritroceratodermia Variável , Ictiose Lamelar , Ictiose , Ceratodermia Palmar e Plantar , Humanos , Eritroceratodermia Variável/genética , Ictiose Lamelar/genética , Ictiose/genética , Mutação , Glucosilceramidas , Transportadores de Cassetes de Ligação de ATP/genéticaRESUMO
Tumors constitute a large class of diseases that affect different organs and cell lineages. The molecular characterization of cancers of a given type has revealed an extraordinary heterogeneity in terms of genetic alterations and DNA mutations; heterogeneity that is further highlighted by single-cell DNA sequencing of individual patients. To address these issues, drugs that specifically target genes or altered pathways in cancer cells are continuously developed. Indeed, the genetic fingerprint of individual tumors can direct the modern therapeutic approaches to selectively hit the tumor cells while sparing the healthy ones. In this context, the concept of precision medicine finds a vast field of application. In this review, we will briefly list some classes of target drugs (Bcl-2 family modulators, Tyrosine Kinase modulators, PARP inhibitors, and growth factors inhibitors) and discuss the application of immunotherapy in tumors (T cell-mediated immunotherapy and CAR-T cells) that in recent years has drastically changed the prognostic outlook of aggressive cancers. We will also consider how apoptosis could represent a primary end point in modern cancer therapy and how "classic" chemotherapeutic drugs that induce apoptosis are still utilized in therapeutic schedules that involve the use of target drugs or immunotherapy to optimize the antitumor response.
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Morte Celular/efeitos dos fármacos , Neoplasias/terapia , Medicina de Precisão/métodos , Apoptose , Genes bcl-2 , Humanos , Imunoterapia , Imunoterapia Adotiva , Peptídeos e Proteínas de Sinalização Intercelular , Neoplasias/genética , Inibidores de Poli(ADP-Ribose) Polimerases , Proteínas Tirosina Quinases/antagonistas & inibidoresRESUMO
BACKGROUND: This study aims to investigate: (a) the putative association between the presence of microcalcifications and the expression of both epithelial-to-mesenchymal transition and bone biomarkers, (b) the role of microcalcifications in the breast osteoblast-like cells (BOLCs) formation, and (c) the association between microcalcification composition and breast cancer progression. METHODS: We collected 174 biopsies on which we performed immunohistochemical and ultrastructural analysis. In vitro experiments were performed to demonstrate the relationship among microcalcification, BOLCs development, and breast cancer occurrence. Ex vivo investigations demonstrated the significant increase of breast osteoblast-like cells in breast lesions with microcalcifications with respect to those without microcalcifications. RESULTS: In vitro data displayed that in the presence of calcium oxalate and activated monocytes, breast cancer cells undergo epithelial to mesenchymal transition. Also, in this condition, cells acquired an osteoblast phenotype, thus producing hydroxyapatite. To further confirm in vitro data, we studied 15 benign lesions with microcalcification from patients that developed a malignant condition in the same breast quadrant. Immunohistochemical analysis showed macrophages' polarization in benign lesions with calcium oxalate. CONCLUSIONS: Altogether, our data shed new light about the role of microcalcifications in breast cancer occurrence and progression.
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Neoplasias da Mama/metabolismo , Calcinose , Transição Epitelial-Mesenquimal , Macrófagos/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , HumanosRESUMO
UNLABELLED: Fulminant hepatitis (FH) is a disease characterized by massive destruction of hepatocytes with severe impairment of liver function. The pathogenesis of FH is not fully understood, but hyperactivity of T cells and macrophages with excessive production of cytokines are important hallmarks of the condition. In this study, we investigated the role of interleukin (IL)-25 in FH. IL-25 expression was evaluated in patients with FH and in livers of mice with FH induced by D-galactosamine (D-Gal) and lipopolysaccharide (LPS). Mice were treated with IL-25 before D-Gal/LPS-induced FH and before or after concanavalin A (ConA)-induced FH. Mononuclear cells were isolated from livers of mice treated with or without IL-25 and analyzed for GR1(+) CD11b(+) cells. CFSE-labeled T cells were cocultured with GR1(+) CD11b(+) cells and their proliferation was evaluated by flow cytometry. Mice were also treated with a depleting anti-GR1 antibody before IL-25 and D-Gal/LPS administration. IL-25 was constitutively expressed in mouse and human liver and down-regulated during FH. IL-25 prevented D-Gal/LPS-induced FH and this effect was associated with increased infiltration of the liver with cells coexpressing GR1 and CD11b. In vitro studies showed that GR1(+) CD11b(+) cells isolated from mice given IL-25 inhibited T-cell proliferation. Consistently, in vivo depletion of GR1(+) cells abrogated the protective effect of IL-25 in experimental D-Gal/LPS-induced FH. IL-25 was both preventive and therapeutic in ConA-induced FH. CONCLUSIONS: IL-25 expression is markedly reduced during human and experimental FH. IL-25 promotes liver accumulation of GR1(+) CD11b(+) cells with immunoregulatory properties.
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Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Hepatócitos/patologia , Interleucinas/uso terapêutico , Células Mieloides/patologia , Linfócitos T/patologia , Animais , Antígeno CD11b/metabolismo , Proliferação de Células/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Técnicas de Cocultura , Concanavalina A/efeitos adversos , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Galactosamina/efeitos adversos , Hepatite/metabolismo , Hepatite/patologia , Hepatócitos/metabolismo , Humanos , Interleucina-17/metabolismo , Interleucinas/metabolismo , Interleucinas/farmacologia , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/metabolismo , Receptores de Quimiocinas/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
The limited availability of hepatic tissue suitable for the treatment of liver disease and drug research encourages the generation of hepatic-like cells from alternative sources as support for the regenerative medicine. Human blood derived stem cells (BDSCs) express surface markers and genes characteristic of pluripotent stem cells and have the ability to differentiate into different cell types, including tissues of endodermal origin (i.e., liver). Therefore they can represent a valuable source of hepatocytes for medicine. In this investigation, we exploited a fast hepatic differentiation protocol to generate hepatocyte-like cells from human BDSCs using only hepatocyte growth factor (HGF) and fibroblast growth factor-4 (FGF-4) as growth factors. The resulting cell population exhibited hepatic cell-like morphology and it was characterized with a variety of biological endpoint analyses. Here, we demonstrate how human BDSCs can be reprogrammed in hepatocyte-like cells by morphological, functional analysis, reverse transcriptase (RT)-PCR, and Western Blot assay. This study defines a fast and easy reprogramming strategy that facilitates the differentiation of human BDSCs along a hepatic lineage and provides a framework for a helpful source in the stem cells therapy and liver disorders.
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Células-Tronco Adultas/fisiologia , Células Sanguíneas/fisiologia , Diferenciação Celular , Linhagem da Célula , Hepatócitos/fisiologia , Células-Tronco Adultas/metabolismo , Biomarcadores/metabolismo , Células Sanguíneas/metabolismo , Western Blotting , Técnicas de Cultura de Células , Diferenciação Celular/genética , Linhagem da Célula/genética , Proliferação de Células , Separação Celular/métodos , Forma Celular , Células Cultivadas , Fator 4 de Crescimento de Fibroblastos/metabolismo , Regulação da Expressão Gênica , Glicogênio/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Hepatócitos/metabolismo , Humanos , Imuno-Histoquímica , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Ureia/metabolismoRESUMO
Stem cell technology has evoked considerable excitement among people interested in the welfare of animals, as it has suggested the potential availability of new tools for several pathologies, including eye disease, which in many cases is considered incurable. One such example is ulcerative keratitis, which is very frequent in horses. Because some of these corneal ulcers can be very severe, progress rapidly and, therefore, can be a possible cause of vision loss, it is important to diagnose them at an early stage and administer an appropriate treatment, which can be medical, surgical, or a combination of both. The therapeutic strategy should eradicate the infection in order to reduce or stop destruction of the cornea. In addition, it should support the corneal structures and control the uveal reaction, and the pain associated with it, in order to minimize scarring. In this study, we address how stem cells derived from peripheral blood can be used also in ophthalmological pathologies. Our results demonstrate that this treatment protocol improved eye disease in four horse cases, including corneal ulcers and one case of retinal detachment. In all cases, we detected a decrease in the intense inflammatory reaction as well as the restoration of the epithelial surface of the central cornea.
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Oftalmopatias/terapia , Oftalmopatias/veterinária , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/veterinária , Doenças dos Cavalos/terapia , Animais , Mordeduras e Picadas/patologia , Mordeduras e Picadas/terapia , Mordeduras e Picadas/veterinária , Úlcera da Córnea/patologia , Úlcera da Córnea/terapia , Úlcera da Córnea/veterinária , Oftalmopatias/patologia , Feminino , Doenças dos Cavalos/patologia , Cavalos , Ceratite/patologia , Ceratite/terapia , Ceratite/veterinária , Masculino , Descolamento Retiniano/patologia , Descolamento Retiniano/terapia , Descolamento Retiniano/veterinária , Uveíte/parasitologia , Uveíte/terapia , Uveíte/veterináriaRESUMO
The translationally controlled tumor protein (TCTP) displays growth-promoting and antiapoptotic properties. To gain information on the role of TCTP in cancer disease, we studied the modulation of TCTP and cell survival under stress conditions on tumor cell lines of different origins. When cancer cells were exposed to a mild oxidative stress, such low doses of Arsenic trioxide (ATO) or hydrogen peroxide (H(2)O(2)), up-regulation of TCTP was observed in cells survived to the treatment. Differently, a strong oxidative hit provided by ATO combined with glutathione (GSH) depletion or condition of glucose deprivation caused a down-modulation of TCTP followed by cell death. Clones with a forced expression of TCTP or with silenced TCTP were obtained from the breast cancer cell line MDA-MB-231. The sensitivity to oxidative stress was strongly enhanced in down-modulated TCTP cells while decreasing in cells with high levels of TCTP. Together these results indicate that TCTP is a survival factor that protects cancer cells from oxidative stress-induced cell-death. We propose TCTP as a "stress hallmark" that may be exploited as a therapeutic target to decrease the resistance of cancer cells to anticancer therapy.
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Biomarcadores Tumorais/metabolismo , Estresse Oxidativo , Trióxido de Arsênio , Arsenicais/farmacologia , Morte Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Glutationa/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Óxidos/farmacologia , Sensibilidade e Especificidade , Relação Estrutura-Atividade , Células Tumorais Cultivadas , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Trastuzumab emtansine (T-DM1) is an anti-human epidermal growth factor receptor 2 (HER2) antibody-drug conjugated to the microtubule-targeting agent emtansine (DM1). T-DM1 is an effective agent in the treatment of patients with HER2-positive breast cancer whose disease has progressed on the first-line trastuzumab containing chemotherapy. However, both primary and acquired tumour resistance limit its efficacy. Increased levels of the phosphorylated form of Translationally Controlled Tumour Protein (phospho-TCTP) have been shown to be associated with a poor clinical response to trastuzumab therapy in HER2-positive breast cancer. Here we show that phospho-TCTP is essential for correct mitosis in human mammary epithelial cells. Reduction of phospho-TCTP levels by dihydroartemisinin (DHA) causes mitotic aberration and increases microtubule density in the trastuzumab-resistant breast cancer cells HCC1954 and HCC1569. Combinatorial studies show that T-DM1 when combined with DHA is more effective in killing breast cells compared to the effect induced by any single agent. In an orthotopic breast cancer xenograft model (HCC1954), the growth of the tumour cells resumes after having achieved a complete response to T-DM1 treatment. Conversely, DHA and T-DM1 treatment induces a severe and irreversible cytotoxic effect, even after treatment interruption, thus, improving the long-term efficacy of T-DM1. These results suggest that DHA increases the effect of T-DM1 as poison for microtubules and supports the clinical development of the combination of DHA and T-DM1 for the treatment of aggressive HER2-overexpressing breast cancer.
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Artemisininas/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos , Microtúbulos/metabolismo , Receptor ErbB-2/metabolismo , Trastuzumab/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA , Feminino , Humanos , Camundongos SCID , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trastuzumab/farmacologia , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
In the frame of the VITA mission of the Italian Space Agency (ASI), we addressed the problem of Space osteoporosis by using human blood-derived stem cells (BDSCs) as a suitable osteogenic differentiation model. In particular, we investigated proteomic and epigenetic changes in BDSCs during osteoblastic differentiation induced by rapamycin under microgravity conditions. A decrease in the expression of 4 embryonic markers (Sox2, Oct3/4, Nanog and E-cadherin) was found to occur to a larger extent on board the ISS than on Earth, along with an earlier activation of the differentiation process towards the osteogenic lineage. The changes in the expression of 4 transcription factors (Otx2, Snail, GATA4 and Sox17) engaged in osteogenesis supported these findings. We then ascertained whether osteogenic differentiation of BDSCs could depend on epigenetic regulation, and interrogated changes of histone H3 that is crucial in this type of gene control. Indeed, we found that H3K4me3, H3K27me2/3, H3K79me2/3 and H3K9me2/3 residues are engaged in cellular reprogramming that drives gene expression. Overall, we suggest that rapamycin induces transcriptional activation of BDSCs towards osteogenic differentiation, through increased GATA4 and Sox17 that modulate downstream transcription factors (like Runx2), critical for bone formation. Additional studies are warranted to ascertain the possible exploitation of these data to identify new biomarkers and therapeutic targets to treat osteoporosis, not only in Space but also on Earth.
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Medicina Aeroespacial , Epigênese Genética , Osteogênese , Osteoporose/fisiopatologia , Proteoma , Ausência de Peso , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem da Célula , Fator de Transcrição GATA4/metabolismo , Histonas/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Osteoporose/genética , Osteoporose/metabolismo , Fatores de Transcrição Otx/metabolismo , Proteômica , Fatores de Transcrição SOXF/metabolismo , Sirolimo/farmacologia , Fatores de Transcrição da Família Snail/metabolismoRESUMO
p53, with its family members p63 and p73, have been shown to promote myoblast differentiation by regulation of the function of the retinoblastoma protein and by direct activation of p21Cip/Waf1 and p57Kip2, promoting cell cycle exit. In previous studies, we have demonstrated that the TAp63γ isoform is the only member of the p53 family that accumulates during in vitro myoblasts differentiation, and that its silencing led to delay in myotube fusion. To better dissect the role of TAp63γ in myoblast physiology, we have generated both sh-p63 and Tet-On inducible TAp63γ clones. Gene array analysis of sh-p63 C2C7 clones showed a significant modulation of genes involved in proliferation and cellular metabolism. Indeed, we found that sh-p63 C2C7 myoblasts present a higher proliferation rate and that, conversely, TAp63γ ectopic expression decreases myoblasts proliferation, indicating that TAp63γ specifically contributes to myoblasts proliferation, independently of p53 and p73. In addition, sh-p63 cells have a defect in mitochondria respiration highlighted by a reduction in spare respiratory capacity and a decrease in complex I, IV protein levels. These results demonstrated that, beside contributing to cell cycle exit, TAp63γ participates to myoblasts metabolism control.
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Mitocôndrias/metabolismo , Mioblastos/metabolismo , Consumo de Oxigênio , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Linhagem Celular , Proliferação de Células , Variações do Número de Cópias de DNA , DNA Mitocondrial , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Subunidades Proteicas , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
The p53 family member p73 controls a wide range of cellular function. Deletion of p73 in mice results in increased tumorigenesis, infertility, neurological defects and altered immune system. Despite the extensive effort directed to define the molecular underlying mechanism of p73 function a clear definition of its transcriptional signature and the extent of overlap with the other p53 family members is still missing. Here we describe a novel TAp73 target, ATP7A a member of a large family of P-type ATPases implicated in human neurogenerative conditions and cancer chemoresistance. Modulation of TAp73 expression influences basal expression level of ATP7A in different cellular models and chromatin immunoprecipitation confirmed a physical direct binding of TAp73 on ATP7A genomic regions. Bioinformatic analysis of expression profile datasets of human lung cancer patients suggests a possible implication of TAp73/ATP7A axis in human cancer. These data provide a novel TAp73-dependent target which might have implications in ageing-related diseases such as cancer and neurodegeneration.
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Envelhecimento/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , ATPases Transportadoras de Cobre/metabolismo , Neoplasias Pulmonares/enzimologia , Proteína Tumoral p73/metabolismo , Fatores Etários , Envelhecimento/genética , Envelhecimento/patologia , Sítios de Ligação , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , ATPases Transportadoras de Cobre/genética , Bases de Dados Genéticas , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Regiões Promotoras Genéticas , Transdução de Sinais , Proteína Tumoral p73/genéticaRESUMO
Abnormal activation of human endogenous retroviruses (HERVs) has been associated with several diseases such as cancer, autoimmunity, and neurological disorders. In particular, in cancer HERV activity and expression have been specifically associated with tumor aggressiveness and patient outcomes. Cancer cell aggressiveness is intimately linked to the acquisition of peculiar plasticity and heterogeneity based on cell stemness features, as well as on the crosstalk between cancer cells and the microenvironment. The latter is a driving factor in the acquisition of aggressive phenotypes, associated with metastasis and resistance to conventional cancer therapies. Remarkably, in different cell types and stages of development, HERV expression is mainly regulated by epigenetic mechanisms and is subjected to a very precise temporal and spatial regulation according to the surrounding microenvironment. Focusing on our research experience with HERV-K involvement in the aggressiveness and plasticity of melanoma cells, this perspective aims to highlight the role of HERV-K in the crosstalk between cancer cells and the tumor microenvironment. The implications for a combination therapy targeted at HERVs with standard approaches are discussed.
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A Pichia pastoris expression system has for the first time been successfully developed to produce rhEPO (recombinant human eosinophil peroxidase). The full-length rhEPO coding sequence was cloned into the pPIC9 vector in frame with the yeast alpha-Factor secretion signal under the transcriptional control of the AOX (acyl-CoA oxidase) promoter, and transformed into P. pastoris strain GS115. Evidence for the production of rhEPO by P. pastoris as a glycosylated dimer precursor of approx. 80 kDa was determined by SDS/PAGE and gel filtration chromatography. Recombinant hEPO undergoes proteolytic processing, similar to that in the native host, to generate two chains of approx. 50 and 20 kDa. A preliminary biochemical characterization of purified rhEPO demonstrated that the spectral and kinetic properties of the recombinant wild-type EPO are comparable with those of the native enzyme and are accompanied by oxidizing activity towards several physiological anionic substrates such as SCN-, Br- and Cl-. On the basis of the estimated K(m) and kcat values it is evident that the pseudohalide SCN- is the most specific substrate for rhEPO, consistent with the catalytic properties of other mammalian EPOs purified from blood.
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Peroxidase de Eosinófilo/genética , Peroxidase de Eosinófilo/metabolismo , Expressão Gênica/genética , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animais , Bioensaio , Cromatografia em Gel , Cromatografia por Troca Iônica , Peroxidase de Eosinófilo/biossíntese , Peroxidase de Eosinófilo/isolamento & purificação , Humanos , Cinética , Peso Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Análise Espectral , Especificidade por Substrato , SuínosRESUMO
BACKGROUND: Melanoma is a heterogeneous tumor in which phenotype-switching and CD133 marker have been associated with metastasis promotion and chemotherapy resistance. CD133 positive (CD133+) subpopulation has also been suggested as putative cancer stem cell (CSC) of melanoma tumor. Human endogenous retrovirus type K (HERV-K) has been described to be aberrantly activated during melanoma progression and implicated in the etiopathogenesis of disease. Earlier, we reported that stress-induced HERV-K activation promotes cell malignant transformation and reduces the immunogenicity of melanoma cells. Herein, we investigated the correlation between HERV-K and the CD133+ melanoma cells during microenvironmental modifications. METHODS: TVM-A12 cell line, isolated in our laboratory from a primary human melanoma lesion, and other commercial melanoma cell lines (G-361, WM-115, WM-266-4 and A375) were grown and maintained in the standard and stem cell media. RNA interference, Real-time PCR, flow cytometry analysis, self-renewal and migration/invasion assays were performed to characterize cell behavior and HERV-K expression. RESULTS: Melanoma cells, exposed to stem cell media, undergo phenotype-switching and expansion of CD133+ melanoma cells, concomitantly promoted by HERV-K activation. Notably, the sorted CD133+ subpopulation showed stemness features, characterized by higher self-renewal ability, embryonic genes expression, migration and invasion capacities compared to the parental cell line. RNA interference-mediated downregulation experiments showed that HERV-K has a decisive role to expand and maintain the CD133+ melanoma subpopulation during microenvironmental modifications. Similarly, non nucleoside reverse transcriptase inhibitors (NNRTIs) efavirenz and nevirapine were effective to restrain the activation of HERV-K in melanoma cells, to antagonize CD133+ subpopulation expansion and to induce selective high level apoptosis in CD133+ cells. CONCLUSIONS: HERV-K activation promotes melanoma cells phenotype-switching and is strictly required to expand and maintain the CD133+ melanoma cells with stemness features in response to microenvironmental modifications.
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Antígeno AC133/metabolismo , Retrovirus Endógenos/fisiologia , Melanoma/virologia , Células-Tronco Neoplásicas/citologia , Ativação Viral , Apoptose , Linhagem Celular Tumoral , Movimento Celular , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica , Humanos , Melanoma/imunologia , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/virologia , Microambiente TumoralAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Neuroblastoma/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Neuroblastoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
The major haemoglobin of the sub-Antarctic fish Eleginops maclovinus was structurally and functionally characterised with the aim to compare molecular environmental adaptations in the O(2)-transport system of sub-Antarctic fishes of the suborder Notothenioidei with those of their high-latitude relatives. Ligand-binding kinetics of the major haemoglobin of E. maclovinus indicated strong stabilisation of the liganded quaternary T state, enhanced in the presence of the physiological allosteric effector ATP, compared to that of high-Antarctic Trematomus bernacchii. The activation enthalpy for O(2) dissociation was dramatically lower than that in T. bernacchii haemoglobin, suggesting remarkable differences in temperature sensitivity and structural changes associated with O(2) release and exit from the protein. The haemoglobin functional properties, together with the X-ray structure of the CO form at 1.49 Å resolution, the first of a temperate notothenioid, strongly support the hypothesis that in E. maclovinus, whose life-style varies according to changes in habitat, the mechanisms that regulate O(2) affinity and the ATP-induced Root effect differ from those of high-Antarctic Notothenioids.
Assuntos
Trifosfato de Adenosina/metabolismo , Hemoglobinas/química , Hemoglobinas/metabolismo , Oxigênio/metabolismo , Perciformes/metabolismo , Adaptação Fisiológica , Animais , Regiões Antárticas , Monóxido de Carbono/metabolismo , Carboxihemoglobina/química , Carboxihemoglobina/metabolismo , Temperatura Baixa , Cristalografia por Raios X , Ecossistema , Cinética , Ligantes , Consumo de Oxigênio , Oxiemoglobinas/química , Oxiemoglobinas/metabolismo , Perciformes/genética , Filogenia , Análise de Sequência de DNA , TermodinâmicaRESUMO
OBJECTIVE: As a better understanding of the molecular basis of carcinogenesis has emerged, oncogene-specific cell-signaling pathways have been successfully targeted to treat human malignances. Despite impressive advances in oncogene-directed therapeutics, genetic instability in cancer cells often manifest acquired resistance. This is particularly noted in the use of tyrosine kinase inhibitors therapies and not more evident than for chronic myeloid leukemia. Therefore, it is of great importance to understand the molecular mechanisms affecting cancer cell sensitivity and resistance to tyrosine kinase inhibitors. MATERIALS AND METHODS: In this study, we used continuous exposure to stepwise increasing concentrations of imatinib (0.6-1 µM) to select imatinib-resistant K562 cells. RESULTS: Expression of BCR-ABL increased both at RNA and protein levels in imatinib-resistant cell lines. Furthermore, expression levels of sphingosine kinase 1 (SphK1) were increased significantly in resistant cells, channeling sphingoid bases to the SphK1 pathway and activating sphingosine-1-phosphate-dependent tyrosine phosphorylation pathways that include the adaptor protein Crk. The partial inhibition of SphK1 activity by N,N-dimethylsphingosine or expression by small interfering RNA increased sensitivity to imatinib-induced apoptosis in resistant cells and returned BCR-ABL to baseline levels. To determine the resistance mechanism-induced SphK1 upregulation, we used pharmacological inhibitors of the phosphoinositide 3-kinase/AKT/mammalian target of rapamycin signaling pathway and observed robust downmodulation of SphK1 expression and activity when AKT2, but not AKT1 or AKT3, was suppressed. CONCLUSIONS: These results demonstrate that SphK1 is upregulated in imatinib-resistant K562 cells by a pathway contingent on a phosphoinositide 3-kinase/AKT2/mammalian target of rapamycin signaling pathway. We propose that SphK1 plays an important role in development of acquired resistance to imatinib in chronic myeloid leukemia cell lines.
Assuntos
Antineoplásicos/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirimidinas/farmacologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Apoptose , Sequência de Bases , Benzamidas , Northern Blotting , Western Blotting , Primers do DNA , Resistencia a Medicamentos Antineoplásicos , Humanos , Mesilato de Imatinib , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para CimaRESUMO
OBJECTIVE: To verify the effect of and to date the unknown T677C mutation of the human N-acetylaspartoacylase (hASPA) gene on the function of the mutated enzyme. DESIGN AND METHODS: Wild type and I226T-mutated proteins were expressed and purified from a transformed Escherichia coli colony. Enzymatic activities were measured in the presence of varying substrate concentrations. RESULTS: Whilst kinetic parameters of wild type hASPA were in line with data in literature, I226T-mutated hASPA showed no enzymatic activity. CONCLUSION: Data indicated that this new mutation might be responsible in homozygosis for the phenotype corresponding to Canavan disease.