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1.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361060

RESUMO

Homeodomain-interacting protein kinase 2 (HIPK2) is a serine-threonine kinase that phosphorylates various transcriptional and chromatin regulators, thus modulating numerous important cellular processes, such as proliferation, apoptosis, DNA damage response, and oxidative stress. The role of HIPK2 in the pathogenesis of cancer and fibrosis is well established, and evidence of its involvement in the homeostasis of multiple organs has been recently emerging. We have previously demonstrated that Hipk2-null (Hipk2-KO) mice present cerebellar alterations associated with psychomotor abnormalities and that the double ablation of HIPK2 and its interactor HMGA1 causes perinatal death due to respiratory failure. To identify other alterations caused by the loss of HIPK2, we performed a systematic morphological analysis of Hipk2-KO mice. Post-mortem examinations and histological analysis revealed that Hipk2 ablation causes neuronal loss, neuronal morphological alterations, and satellitosis throughout the whole central nervous system (CNS); a myopathic phenotype characterized by variable fiber size, mitochondrial proliferation, sarcoplasmic inclusions, morphological alterations at neuromuscular junctions; and a cardiac phenotype characterized by fibrosis and cardiomyocyte hypertrophy. These data demonstrate the importance of HIPK2 in the physiology of skeletal and cardiac muscles and of different parts of the CNS, thus suggesting its potential relevance for different new aspects of human pathology.


Assuntos
Sistema Nervoso Central/patologia , Fibrose/patologia , Miocárdio/patologia , Neurônios/patologia , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Sistema Nervoso Central/metabolismo , Feminino , Fibrose/metabolismo , Proteínas HMGA/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miocárdio/metabolismo , Neurônios/metabolismo , Fenótipo , Fosforilação
2.
Biochem J ; 477(8): 1363-1366, 2020 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-32322896

RESUMO

Cell-penetrating peptides (CPPs) are short peptides able to cross the cellular membranes without any interaction with specific receptors. Thanks to their ability to transport various cargo inside the cells are emerged as powerful therapeutic agents alternative to small molecules. In recent years, numerous preclinical studies provided promising results for the treatment of various human diseases. Several CPP-conjugated compounds are under clinical trials.


Assuntos
Peptídeos Penetradores de Células/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/administração & dosagem , Peptídeos Penetradores de Células/química , Tratamento Farmacológico , Humanos
3.
Mol Med Rep ; 21(3): 1501-1508, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32016459

RESUMO

Colorectal cancer (CRC) is the third most prevalent type of cancer worldwide. It is also the second most common cause of cancer­associated mortality; it accounted for about 9.2% of all cancer deaths in 2018, most of which were due to resistance to therapy. The main treatment for CRC is surgery, generally associated with chemotherapy, radiation therapy and combination therapy. However, while chemo­radiotherapy kills differentiated cancer cells, mesenchymal stem­like cells are resistant to this treatment, and this can give rise to therapy­resistant tumors. Our previous study isolated T88 primary colon cancer cells from a patient with sporadic colon cancer. These cells exhibited mesenchymal and epithelial features, high levels of epithelial­to­mesenchymal transition transcription factors, and stemness markers. In addition, it was revealed that lithium chloride (LiCl), a specific glycogen synthase kinase (GSK)­3ß inhibitor, induced both the mesenchymal­to­epithelial transition and differentiation, and also reduced cell migration, stemness features and cell plasticity in these primary colon cancer cells. The aim of the present study was to investigate the effect of LiCl treatment on the viability of primary colon cancer cells exposed to 7 Gy delivered by high­energy photon beams, which corresponds to 6 megavolts of energy. To achieve this aim, the viability of irradiated T88 cells was compared with that of irradiated T88 cells pre­treated with LiCl. As expected, it was observed that LiCl sensitized primary colon cancer cells to high­energy photon irradiation treatment. Notably, the decrease in cell viability was greater with combined therapy than with irradiation alone. To explore the molecular basis of this response, the effect of LiCl on the expression of Bax, p53 and Survivin, which are proteins involved in the apoptotic mechanism and in death escape, was analyzed. The present study revealed that LiCl upregulated the expression of pro­apoptotic proteins and downregulated the expression of proteins involved in survival. These effects were enhanced by high­energy photon irradiation, suggesting that LiCl could be used to sensitize colon cancer cells to radiation therapy.


Assuntos
Cloreto de Lítio/farmacologia , Fótons , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/diagnóstico , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/radioterapia , Humanos , Radioterapia de Alta Energia/métodos , Transdução de Sinais/efeitos dos fármacos
4.
Cell Death Dis ; 10(10): 747, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582725

RESUMO

The serine-threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) modulates important cellular functions during development, acting as a signal integrator of a wide variety of stress signals, and as a regulator of transcription factors and cofactors. We have previously demonstrated that HIPK2 binds and phosphorylates High-Mobility Group A1 (HMGA1), an architectural chromatinic protein ubiquitously expressed in embryonic tissues, decreasing its binding affinity to DNA. To better define the functional role of HIPK2 and HMGA1 interaction in vivo, we generated mice in which both genes are disrupted. About 50% of these Hmga1/Hipk2 double knock-out (DKO) mice die within 12 h of life (P1) for respiratory failure. The DKO mice present an altered lung morphology, likely owing to a drastic reduction in the expression of surfactant proteins, that are required for lung development. Consistently, we report that both HMGA1 and HIPK2 proteins positively regulate the transcriptional activity of the genes encoding the surfactant proteins. Moreover, these mice display an altered expression of thyroid differentiation markers, reasonably because of a drastic reduction in the expression of the thyroid-specific transcription factors PAX8 and FOXE1, which we demonstrate here to be positively regulated by HMGA1 and HIPK2. Therefore, these data indicate a critical role of HIPK2/HMGA1 cooperation in lung and thyroid development and function, suggesting the potential involvement of their impairment in the pathogenesis of human lung and thyroid diseases.


Assuntos
Proteína HMGA1a/genética , Proteínas Serina-Treonina Quinases/genética , Doenças Respiratórias/genética , Glândula Tireoide/anormalidades , Animais , Animais Recém-Nascidos , Desenvolvimento Embrionário , Deleção de Genes , Regulação da Expressão Gênica , Proteína HMGA1a/metabolismo , Células HeLa , Humanos , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Associadas a Surfactantes Pulmonares , Doenças Respiratórias/patologia , Glândula Tireoide/embriologia , Glândula Tireoide/patologia
5.
Front Neurosci ; 13: 673, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31316342

RESUMO

PARK20, an early onset autosomal recessive parkinsonism is due to mutations in the phosphatidylinositol-phosphatase Synaptojanin 1 (Synj1). We have recently shown that the early endosomal compartments are profoundly altered in PARK20 fibroblasts as well as the endosomal trafficking. Here, we report that PARK20 fibroblasts also display a drastic alteration of the architecture and function of the early secretory compartments. Our results show that the exit machinery from the Endoplasmic Reticulum (ER) and the ER-to-Golgi trafficking are markedly compromised in patient cells. As a consequence, PARK20 fibroblasts accumulate large amounts of cargo proteins within the ER, leading to the induction of ER stress. Interestingly, this stressful state is coupled to the activation of the PERK/eIF2α/ATF4/CHOP pathway of the Unfolded Protein Response (UPR). In addition, PARK20 fibroblasts reveal upregulation of oxidative stress markers and total ROS production with concomitant alteration of the morphology of the mitochondrial network. Interestingly, treatment of PARK20 cells with GSK2606414 (GSK), a specific inhibitor of PERK activity, restores the level of ROS, signaling a direct correlation between ER stress and the induction of oxidative stress in the PARK20 cells. All together, these findings suggest that dysfunction of early secretory pathway might contribute to the pathogenesis of the disease.

6.
Int J Mol Sci ; 20(11)2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31141937

RESUMO

Acute administration of a high level of extracellular citrate displays an anti-proliferative effect on both in vitro and in vivo models. However, the long-term effect of citrate treatment has not been investigated yet. Here, we address this question in PC3 cells, a prostate-cancer-derived cell line. Acute administration of high levels of extracellular citrate impaired cell adhesion and inhibited the proliferation of PC3 cells, but surviving cells adapted to grow in the chronic presence of 20 mM citrate. Citrate-resistant PC3 cells are significantly less glycolytic than control cells. Moreover, they overexpress short-form, citrate-insensitive phosphofructokinase 1 (PFK1) together with full-length PFK1. In addition, they show traits of mesenchymal-epithelial transition: an increase in E-cadherin and a decrease in vimentin. In comparison with PC3 cells, citrate-resistant cells display morphological changes that involve both microtubule and microfilament organization. This was accompanied by changes in homeostasis and the organization of intracellular organelles. Thus, the mitochondrial network appears fragmented, the Golgi complex is scattered, and the lysosomal compartment is enlarged. Interestingly, citrate-resistant cells produce less total ROS but accumulate more mitochondrial ROS than control cells. Consistently, in citrate-resistant cells, the autophagic pathway is upregulated, possibly sustaining their survival. In conclusion, chronic administration of citrate might select resistant cells, which could jeopardize the benefits of citrate anticancer treatment.


Assuntos
Citratos/farmacologia , Neoplasias da Próstata/metabolismo , Autofagia/efeitos dos fármacos , Caderinas/metabolismo , Proliferação de Células/efeitos dos fármacos , Glicólise , Humanos , Masculino , Microtúbulos/metabolismo , Células PC-3 , Fosfofrutoquinase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Vimentina/metabolismo
8.
Cell Physiol Biochem ; 47(5): 1951-1976, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29969760

RESUMO

A general hallmark of neurological diseases is the loss of redox homeostasis that triggers oxidative damages to biomolecules compromising neuronal function. Under physiological conditions the steady-state concentrations of reactive oxygen species (ROS) and reactive nitrogen species (RNS) are finely regulated for proper cellular functions. Reduced surveillance of endogenous antioxidant defenses and/or increased ROS/RNS production leads to oxidative stress with consequent alteration of physiological processes. Neuronal cells are particularly susceptible to ROS/RNS due to their biochemical composition. Overwhelming evidences indicate that nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-linked pathways are involved in protective mechanisms against oxidative stress by regulating antioxidant and phase II detoxifying genes. As such, Nrf2 deregulation has been linked to both aging and pathogenesis of many human chronic diseases, including neurodegenerative ones such as Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Nrf2 activity is tightly regulated by a fine balance between positive and negative modulators. A better understanding of the regulatory mechanisms underlying Nrf2 activity could help to develop novel therapeutic interventions to prevent, slow down or possibly reverse various pathological states. To this end, microRNAs (miRs) are attractive candidates because they are linked to intracellular redox status being regulated and, post-transcriptionally, regulating key components of ROS/RNS pathways, including Nrf2.


Assuntos
Envelhecimento/metabolismo , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Doenças Neurodegenerativas/metabolismo , Estresse Oxidativo , Transdução de Sinais , Envelhecimento/genética , Envelhecimento/patologia , Animais , Humanos , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia
9.
Microrna ; 7(3): 178-186, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29793420

RESUMO

The Homeodomain-Interacting Protein Kinases (HIPKs) HIPK1, HIPK2 and HIPK3 are Ser/Thr kinases which interact with homeobox proteins and other transcription factors, acting as transcriptional coactivators or corepressors. HIPKs contribute to regulate several biological processes, such as signal transduction, apoptosis, embryonic development, DNA-damage response, and cellular proliferation, in response to various extracellular stimuli. Recently it has emerged that, in addition to their role in cancer, fibrosis and diabetes, HIPKs may also be involved in other human diseases, including Amyotrophic Lateral Sclerosis (ALS), Rett syndrome, cerebellar diseases, and retinal vascular dysfunction. METHODS: Here, we update our previous paper concerning the regulation of HIPK proteins expression by microRNAs (miRNAs), pointing out the most recent findings about new cellular mechanisms and diseases which are affected by the interplay between HIPKs and miRNAs. CONCLUSION: Recently, it has emerged that HIPKs and their related miRNAs are involved in diabetic nephropathy, gastric cancer chemoresistance, cervical cancer progression, and recombinant protein expression in cultured cells. Interestingly, circular RNAs (circRNAs) deriving from HIPK2 and HIPK3 loci also modulate cellular proliferation and viability by sponging several miRNAs, thus emerging as new putative therapeutic targets for diabetes-associated retinal vascular dysfunction, astrogliosis and cancer.


Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , MicroRNAs/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinases/metabolismo , Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais
11.
Cell Death Dis ; 9(3): 385, 2018 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-29515184

RESUMO

Recently, a new form of autosomal recessive early-onset parkinsonism (PARK20), due to mutations in the gene encoding the phosphoinositide phosphatase, Synaptojanin 1 (Synj1), has been reported. Several genes responsible for hereditary forms of Parkinson's disease are implicated in distinct steps of the endolysosomal pathway. However, the nature and the degree of endocytic membrane trafficking impairment in early-onset parkinsonism remains elusive. Here, we show that depletion of Synj1 causes drastic alterations of early endosomes, which become enlarged and more numerous, while it does not affect the morphology of late endosomes both in non-neuronal and neuronal cells. Moreover, Synj1 loss impairs the recycling of transferrin, while it does not alter the trafficking of the epidermal growth factor receptor. The ectopic expression of Synj1 restores the functions of early endosomes, and rescues these trafficking defects in depleted cells. Importantly, the same alterations of early endosomal compartments and trafficking defects occur in fibroblasts of PARK20 patients. Our data indicate that Synj1 plays a crucial role in regulating the homeostasis and functions of early endosomal compartments in different cell types, and highlight defective cellular pathways in PARK20. In addition, they strengthen the link between endosomal trafficking and Parkinson's disease.


Assuntos
Endossomos/metabolismo , Fibroblastos/metabolismo , Doença de Parkinson/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Western Blotting , Linhagem Celular , Células Cultivadas , Células HeLa , Humanos , Microscopia de Fluorescência , Mutação/genética , Doença de Parkinson/genética , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
13.
Cell Death Differ ; 24(11): 1948-1962, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28777374

RESUMO

High Mobility Group A1 (HMGA1) is an architectural chromatin protein whose overexpression is a feature of malignant neoplasias with a causal role in cancer initiation and progression. HMGA1 promotes tumor growth by several mechanisms, including increase of cell proliferation and survival, impairment of DNA repair and induction of chromosome instability. Autophagy is a self-degradative process that, by providing energy sources and removing damaged organelles and misfolded proteins, allows cell survival under stress conditions. On the other hand, hyper-activated autophagy can lead to non-apoptotic programmed cell death. Autophagy deregulation is a common feature of cancer cells in which has a complex role, showing either an oncogenic or tumor suppressor activity, depending on cellular context and tumor stage. Here, we report that depletion of HMGA1 perturbs autophagy by different mechanisms. HMGA1-knockdown increases autophagosome formation by constraining the activity of the mTOR pathway, a major regulator of autophagy, and transcriptionally upregulating the autophagy-initiating kinase Unc-51-like kinase 1 (ULK1). Consistently, functional experiments demonstrate that HMGA1 binds ULK1 promoter region and negatively regulates its transcription. On the other hand, the increase in autophagosomes is not associated to a proportionate increase in their maturation. Overall, the effects of HMGA1 depletion on autophagy are associated to a decrease in cell proliferation and ultimately impact on cancer cells viability. Importantly, silencing of ULK1 prevents the effects of HMGA1-knockdown on cellular proliferation, viability and autophagic activity, highlighting how these effects are, at least in part, mediated by ULK1. Interestingly, this phenomenon is not restricted to skin cancer cells, as similar results have been observed also in HeLa cells silenced for HMGA1. Taken together, these results clearly indicate HMGA1 as a key regulator of the autophagic pathway in cancer cells, thus suggesting a novel mechanism through which HMGA1 can contribute to cancer progression.


Assuntos
Autofagia , Proteína HMGA1a/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Proliferação de Células , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Inativação Gênica , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Transcrição Genética
14.
Int J Mol Sci ; 17(9)2016 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-27649143

RESUMO

Resveratrol, a dietary polyphenol, is under consideration as chemopreventive and chemotherapeutic agent for several diseases, including cancer. However, its mechanisms of action and its effects on non-tumor cells, fundamental to understand its real efficacy as chemopreventive agent, remain largely unknown. Proline-rich tyrosine kinase 2 (PYK2), a non-receptor tyrosine kinase acting as signaling mediator of different stimuli, behaves as tumor-suppressor in prostate. Since, PYK2 and RSV share several fields of interaction, including oxidative stress, we have investigated their functional relationship in human non-transformed prostate EPN cells and in their tumor-prone counterpart EPN-PKM, expressing a PYK2 dead-kinase mutant. We show that RSV has a strong biological activity in both cell lines, decreasing ROS production, inducing morphological changes and reversible growth arrest, and activating autophagy but not apoptosis. Interestingly, the PYK2 mutant increases basal ROS and autophagy levels, and modulates the intensity of RSV effects. In particular, the anti-oxidant effect of RSV is more potent in EPN than in EPN-PKM, whereas its anti-proliferative and pro-autophagic effects are more significant in EPN-PKM. Consistently, PYK2 depletion by RNAi replicates the effects of the PKM mutant. Taken together, our results reveal that PYK2 and RSV act on common cellular pathways and suggest that RSV effects on prostate cells may depend on mutational-state or expression levels of PYK2 that emerges as a possible mediator of RSV mechanisms of action. Moreover, the observation that resveratrol effects are reversible and not associated to apoptosis in tumor-prone EPN-PKM cells suggests caution for its use in humans.


Assuntos
Antioxidantes/farmacocinética , Quinase 2 de Adesão Focal/genética , Próstata/efeitos dos fármacos , Próstata/metabolismo , Estilbenos/farmacologia , Autofagia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Quinase 2 de Adesão Focal/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Mutação , Estresse Oxidativo/efeitos dos fármacos , Próstata/citologia , Resveratrol
15.
Virology ; 496: 1-8, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27236740

RESUMO

Felis catus papillomavirus type 2 (FcaPV2) DNA is found in feline cutaneous squamous cell carcinomas (SCCs); however, its biological properties are still uncharacterized. In this study, we successfully expressed FcaPV2 E6 and E7 putative oncogenes in feline epithelial cells and demonstrated that FcaPV2 E6 binds to p53, impairing its protein level. In addition, E6 and E7 inhibited ultraviolet B (UVB)-triggered accumulation of p53, p21 and pro-apoptotic markers such as Cleaved Caspase3, Bax and Bak, suggesting a synergistic action of the virus with UV exposure in tumour pathogenesis. Furthermore, FcaPV2 E7 bound to feline pRb and impaired pRb levels, resulting in upregulation of the downstream pro-proliferative genes Cyclin A and Cdc2. Importantly, we demonstrated mRNA expression of FcaPV2 E2, E6 and E7 in feline SCC samples, strengthening the hypothesis of a causative role in the development of feline SCC.


Assuntos
Carcinoma de Células Escamosas/etiologia , Regulação Viral da Expressão Gênica , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Papillomaviridae/fisiologia , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Ativação Transcricional , Animais , Proteínas de Transporte , Gatos , Linhagem Celular , Camundongos , Infecções por Papillomavirus/veterinária , Ligação Proteica , RNA Mensageiro/genética , Transdução de Sinais
16.
BMC Biol ; 14: 24, 2016 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-27036552

RESUMO

BACKGROUND: A crucial event in the differentiation of mouse embryonic stem cells (ESCs) is the exit from the pluripotent ground state that leads to the acquisition of the 'primed' pluripotent phenotype, characteristic of the epiblast-like stem cells (EpiLCs). The transcription factors Oct4 and Otx2 play a key role in this phenomenon. In particular, Otx2 pioneers and activates new enhancers, which are silent in ESCs and which control the transcription of genes responsible for the acquisition of the EpiLC phenotype. An important point that remains to be addressed is the mechanism through which Otx2 engages the new enhancers and stably associates with them. Hmga2 is a member of the high-mobility group family of proteins, non-histone components of chromatin whose expression is high during embryogenesis and becomes low or undetectable in adults. Its high expression during embryogenesis suggests that Hmga2 fulfills important roles in development. RESULTS: Here, we demonstrate that Hmga2 accumulates soon after the induction of ESC differentiation. Its suppression hampers the exit of ESCs from the pluripotent ground state and their differentiation into EpiLCs. Mechanistically, Hmga2 controls the differentiation process by cooperating with Otx2 in the pioneering of new enhancers. In Hmga2 null induced pluripotent stem cells we observe that Otx2 fails to regulate its target genes upon the induction of differentiation. Hmga2 associates to Otx2-bound loci in EpiLCs, and in Hmga2 KO cells Otx2 is unable to engage and activate the new enhancers, thus indicating that Hmga2 is required for the binding of Otx2 to its cis-elements. We find that this mechanism also operates on the Hmga2 gene, which is one of the targets of Otx2, thus indicating the existence of a positive feedback loop. CONCLUSIONS: Our findings reveal a novel mechanism necessary for the exit of ESCs from the pluripotent ground state. Upon the induction of ESC differentiation, Otx2 alone or in combination with Oct4 engages new enhancers, which are silent in undifferentiated ESCs. The Hmga2 gene is activated by Otx2 and Hmga2 protein binds to the enhancers targeted by Otx2, thus facilitating the engagement and/or the stable association of Otx2. Therefore, our results demonstrate that Hmga2 is a key element of the regulatory network that governs the exit of ESCs from the pluripotent ground state.


Assuntos
Células-Tronco Embrionárias/citologia , Regulação da Expressão Gênica , Proteína HMGA2/genética , Fatores de Transcrição Otx/metabolismo , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Deleção de Genes , Proteína HMGA2/metabolismo , Camundongos , Células-Tronco Pluripotentes/metabolismo
17.
Cell Cycle ; 15(6): 812-8, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26889953

RESUMO

The High Mobility Group A1 proteins (HMGA1) are nonhistone chromatinic proteins with a critical role in development and cancer. We have recently reported that HMGA1 proteins are able to increase the expression of spindle assembly checkpoint (SAC) genes, thus impairing SAC function and causing chromosomal instability in cancer cells. Moreover, we found a significant correlation between HMGA1 and SAC genes expression in human colon carcinomas. Here, we report that mouse embryonic fibroblasts null for the Hmga1 gene show downregulation of Bub1, Bub1b, Mad2l1 and Ttk SAC genes, and present several features of chromosomal instability, such as nuclear abnormalities, binucleation, micronuclei and karyotypic alterations. Interestingky, also MEFs carrying only one impaired Hmga1 allele present karyotypic alterations. These results indicate that HMGA1 proteins regulate SAC genes expression and, thereby, genomic stability also in embryonic cells.


Assuntos
Instabilidade Cromossômica , Fibroblastos/metabolismo , Pontos de Checagem da Fase G2 do Ciclo Celular , Proteína HMGA1a/genética , Pontos de Checagem da Fase M do Ciclo Celular/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Núcleo Celular/patologia , Embrião de Mamíferos , Fibroblastos/patologia , Regulação da Expressão Gênica , Teste de Complementação Genética , Proteína HMGA1a/deficiência , Cariótipo , Camundongos , Camundongos Knockout , Micronúcleos com Defeito Cromossômico , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais
18.
Microrna ; 4(3): 148-57, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26428079

RESUMO

INTRODUCTION: The homeodomain-interacting protein kinase (HIPK) family consists of four evolutionarily conserved and highly related nuclear serine/threonine kinases of recent discovery. They interact with homeobox proteins and other transcription factors, as well as transcriptional coactivators or corepressors depending on the cellular context. HIPK proteins are sensors for various extracellular stimuli, which control key cellular functions such as signal transduction to downstream effectors that regulate apoptosis, embryonic development, DNA-damage response, and cellular proliferation. Thus, HIPKs are involved in proliferative diseases such as cancer and fibrosis. mRNA levels and protein stability tightly regulate expression levels of HIPKs. METHODS: Here, we review recent works investigating the regulation of HIPKs expression by microRNAs (miRNAs) that are involved in the control of cell proliferation, sensitivity to chemotherapeutic drugs, epithelial-mesenchymal transition, and glucose-stimulated insulin secretion. CONCLUSION: It appears that HIPK family members, and their related miRNAs, may be considered as novel therapeutic targets for treating cancer, renal fibrosis and type 2 diabetes.


Assuntos
Núcleo Celular/metabolismo , MicroRNAs/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Apoptose , Núcleo Celular/genética , Núcleo Celular/patologia , Dano ao DNA , Desenvolvimento Embrionário/genética , Transição Epitelial-Mesenquimal/genética , Fibrose/genética , Fibrose/metabolismo , Humanos , MicroRNAs/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Proteínas Serina-Treonina Quinases/genética , RNA Neoplásico/genética , RNA Neoplásico/metabolismo
19.
Int J Mol Sci ; 16(9): 20375-91, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26343643

RESUMO

Photofrin/photodynamic therapy (PDT) at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53(+/+)) and H1299 (p53(-/-)) cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones) with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.


Assuntos
Apoptose , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Antineoplásicos/farmacologia , Bortezomib/farmacologia , Linhagem Celular Tumoral , Éter de Diematoporfirina/metabolismo , Humanos , Espaço Intracelular/metabolismo , Potencial da Membrana Mitocondrial , Microscopia Confocal , Fotoquimioterapia , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/farmacologia
20.
Int J Mol Sci ; 16(9): 20417-30, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26343645

RESUMO

Although photodynamic therapy (PDT), a therapeutic approach that involves a photosensitizer, light and O2, has been principally considered for the treatment of specific types of cancers, other applications exist, including the treatment of infections. Unfortunately, PDT does not always guarantee full success since it exerts lethal effects only in cells that have taken up a sufficient amount of photosensitizer and have been exposed to adequate light doses, conditions that are not always achieved. Based on our previous experience on the combination PDT/chemotherapy, we have explored the possibility of fighting bacteria that commonly crowd infected surfaces by combining PDT with an antibiotic, which normally does not harm the strain at low concentrations. To this purpose, we employed 5-aminolevulinic acid (5-ALA), a pro-drug that, once absorbed by proliferating bacteria, is converted into the natural photosensitizer Protoporphyrin IX (PpIX), followed by Gentamicin. Photoactivation generates reactive oxygen species (ROS) which damage or kill the cell, while Gentamicin, even at low doses, ends the work. Our experiments, in combination, have been highly successful against biofilms produced by several Gram positive bacteria (i.e., Staphylococcus aureus, Staphylococcus epidermidis, etc.). This original approach points to potentially new and wide applications in the therapy of infections of superficial wounds and sores.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana , Luz , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Gentamicinas/farmacologia , Testes de Sensibilidade Microbiana , Microscopia Confocal
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