RESUMO
BACKGROUND: The COVID-19 pandemic is placing blood and tissue establishments under unprecedented stress, putting its capacity to provide the adequate care needed at risk. Here we reflect on how our integrated organisational model has faced the first impact of the pandemic and describe what challenges, opportunities and lessons have emerged. MATERIALS AND METHODS: The organisational model of the Catalan Blood and Tissue Bank (Banc de Sang i Teixits, BST) is described. The new scenario was managed by following international recommendations and considering the pandemic in a context of volatility, uncertainty, complexity, and ambiguity (VUCA), allowing rapid measures to be taken. These aimed to: ensure donor safety, promote proper responses to patients' needs, ensure the health and well-being of personnel, and prepare for future scenarios. RESULTS: The BST has adapted its activities to the changes in demand. No shortage of any product or service occurred. Donor acceptance, safety and wellbeing were maintained except for tissue donation, which almost completely stopped. To support the health system, several activities have been promoted: large-scale convalescent plasma (CP) production, clinical trials with CP and mesenchymal stromal cells, massive COVID-19 diagnoses, and participation in co-operative research and publications. Haemovigilance is running smoothly and no adverse effects have been detected among donors or patients. DISCUSSION: Several elements have proven to be critical when addressing the pandemic scenario: a) the early creation of a crisis committee in combination with technical recommendations and the recognition of a VUCA scenario; b) identification of the strategies described; c) the integrated donor-to-patient organisational model; d) active Research and Development (R&D); and e) the flexibility of the staff. It is essential to underline the importance of the need for centralised management, effective contingency strategies, and early collaboration with peers.
Assuntos
Bancos de Sangue/organização & administração , COVID-19/epidemiologia , Pandemias , SARS-CoV-2 , Bancos de Tecidos/organização & administração , Bancos de Sangue/provisão & distribuição , Transfusão de Componentes Sanguíneos/estatística & dados numéricos , Doadores de Sangue , Transplante de Medula Óssea , COVID-19/prevenção & controle , COVID-19/terapia , Humanos , Imunização Passiva , Modelos Organizacionais , Doenças Profissionais/prevenção & controle , Segurança , Espanha , Obtenção de Tecidos e Órgãos , Soroterapia para COVID-19RESUMO
RhoE is a small GTPase involved in the regulation of actin cytoskeleton dynamics, cell cycle and apoptosis. The role of RhoE in cancer is currently controversial, with reports of both oncogenic and tumor-suppressive functions for RhoE. Using RhoE-deficient mice, we show here that the absence of RhoE blunts contact-inhibition of growth by inhibiting p27Kip1 nuclear translocation and cooperates in oncogenic transformation of mouse primary fibroblasts. Heterozygous RhoE+/gt mice are more susceptible to chemically induced skin tumors and RhoE knock-down results in increased metastatic potential of cancer cells. These results indicate that RhoE plays a role in suppressing tumor initiation and progression.
Assuntos
Transformação Celular Neoplásica/metabolismo , Inibição de Contato/fisiologia , Neoplasias Experimentais/patologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Western Blotting , Transformação Celular Neoplásica/patologia , Progressão da Doença , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Camundongos Nus , Neoplasias Experimentais/metabolismoRESUMO
RhoE/Rnd3 is an atypical member of the Rho family of small GTPases. In addition to regulating actin cytoskeleton dynamics, RhoE is involved in the regulation of cell proliferation, survival, and metastasis. We examined RhoE expression levels during cell cycle and investigated mechanisms controlling them. We show that RhoE accumulates during G1, in contact-inhibited cells, and when the Akt pathway is inhibited. Conversely, RhoE levels rapidly decrease at the G1/S transition and remain low for most of the cell cycle. We also show that the half-life of RhoE is shorter than that of other Rho proteins and that its expression levels are regulated by proteasomal degradation. The expression patterns of RhoE overlap with that of the cell cycle inhibitor p27. Consistently with an involvement of RhoE in cell cycle regulation, RhoE and p27 levels decrease after overexpression of the F-box protein Skp2. We have identified a region between amino acids 231 and 240 of RhoE as the Skp2-interacting domain and Lys(235) as the substrate for ubiquitylation. Based on our results, we propose a mechanism according to which proteasomal degradation of RhoE by Skp2 regulates its protein levels to control cellular proliferation.
Assuntos
Fase G1/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Quinases Associadas a Fase S/biossíntese , Ubiquitinação/fisiologia , Proteínas rho de Ligação ao GTP/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação da Expressão Gênica/fisiologia , Células HeLa , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Proteínas Quinases Associadas a Fase S/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Neurite formation involves coordinated changes between the actin cytoskeleton and the microtubule network. Rho GTPases are clearly implicated in several aspects of neuronal development and function. Indeed, RhoA is a negative regulator of neurite outgrowth and its effector Rho-kinase mediates the Rho-driven neurite retraction. Considering that RhoE/round protein (Rnd3) acts antagonistically to RhoA and it is also able to bind and inhibit rho kinase-I (p160ROCK) - ROCK-I, it is tempting to speculate a role of RhoE in neurite formation. We show for the first time that, in the absence of nerve growth factor (NGF), RhoE induces neurite-like outgrowth. Our results demonstrate that over-expression of RhoE decreases the activity of RhoA and reduces the expression of both ROCK-I and the phosphorylated myosin light chain phosphatase (MLCPp). Conversely, over-expression of either active RhoA or ROCK-I abolishes the RhoE-promoted neurite outgrowth, suggesting that RhoE induces neurite-like formation through inhibition of the RhoA/ROCK-I signalling. We also show that Rac and Cdc42 have a role in RhoE-induced neurite outgrowth. Finally, the present data further indicate that RhoE may be involved in the NGF-induced neurite outgrowth in PC12 cells, as depletion of RhoE by siRNA reduces the neurite formation induced by NGF. These findings provide new insights into the molecular mechanism implicated in neuronal development and may provide novel therapeutic targets in neurodegenerative disorders.
Assuntos
Neuritos/fisiologia , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Animais , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Fluorescência Verde/genética , Fator de Crescimento Neural/farmacologia , Neuritos/efeitos dos fármacos , Proteínas de Neurofilamentos/metabolismo , Células PC12/citologia , Células PC12/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção/métodos , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Rho GTPases are important regulators of actin cytoskeleton, but they are also involved in cell proliferation, transformation and oncogenesis. One of this proteins, RhoE, inhibits cell proliferation, however the mechanism that regulates this effect remains poorly understood. Therefore, we undertook the present study to determine the role of RhoE in the regulation of cell proliferation. For this purpose we generated an adenovirus system to overexpress RhoE in U87 glioblastoma cells. Our results show that RhoE disrupts actin cytoskeleton organization and inhibits U87 glioblastoma cell proliferation. Importantly, RhoE expressing cells show a reduction in Rb phosphorylation and in cyclin D1 expression. Furthermore, RhoE inhibits ERK activation following serum stimulation of quiescent cells. Based in these findings, we propose that RhoE inhibits ERK activation, thereby decreasing cyclin D1 expression and leading to a reduction in Rb inactivation, and that this mechanism is involved in the RhoE-induced cell growth inhibition. Moreover, we also demonstrate that RhoE induces apoptosis in U87 cells and also in colon carcinoma and melanoma cells. These results indicate that RhoE plays an important role in the regulation of cell proliferation and survival, and suggest that this protein may be considered as an oncosupressor since it is capable to induce apoptosis in several tumor cell lines.
Assuntos
Proliferação de Células , Glioblastoma/patologia , Proteína do Retinoblastoma/metabolismo , Proteínas rho de Ligação ao GTP/fisiologia , Citoesqueleto de Actina/química , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Ciclina D1/metabolismo , Citoesqueleto/química , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Transfecção , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Anoikis is a programmed cell death induced by loss of anchorage that is involved in tissue homeostasis and disease. Ethanol is an important teratogen that induces marked central nervous system (CNS) dysfunctions. Here we show that astrocytes exposed to ethanol undergo morphological changes associated with anoikis, including the peripheral reorganization of both focal adhesions and actin-myosin system, cell contraction, membrane blebbing and chromatin condensation. We found that either the small GTPase RhoA or its effector ROCK-I (Rho kinase), promotes membrane blebbing in astrocytes. Ethanol induces a ROCK-I activation that is mediated by RhoA, rather than by caspase-3 cleavage. Accordingly, the RhoA inhibitor C3, completely abolishes the ethanol-induced ROCK-I activation. Furthermore, inhibition of both RhoA and ROCK prevents the membrane blebbing induced by ethanol. Ethanol also promotes myosin light chain (MLC) phosphorylation, which might be involved in the actin-myosin contraction. All of these findings strongly support that ethanol-exposed astrocytes undergo apoptosis by anoikis and also that the RhoA/ROCK-I/MLC pathway participates in this process.
Assuntos
Anoikis/fisiologia , Astrócitos/efeitos dos fármacos , Astrócitos/fisiologia , Etanol/farmacologia , Cadeias Leves de Miosina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Anoikis/efeitos dos fármacos , Astrócitos/citologia , Caspase 3 , Caspases/metabolismo , Forma Celular , Células Cultivadas , Ativação Enzimática , Peptídeos e Proteínas de Sinalização Intracelular , Cadeias Leves de Miosina/genética , Miosinas/metabolismo , Proteínas Serina-Treonina Quinases/genética , Ratos , Quinases Associadas a rho , Proteína rhoA de Ligação ao GTP/genéticaRESUMO
Rho GTPases are major regulators of cytoskeletal dynamics, but they also affect cell proliferation, transformation, and oncogenesis. RhoE, a member of the Rnd subfamily that does not detectably hydrolyze GTP, inhibits RhoA/ROCK signaling to promote actin stress fiber and focal adhesion disassembly. We have generated fibroblasts with inducible RhoE expression to investigate the role of RhoE in cell proliferation. RhoE expression induced a loss of stress fibers and cell rounding, but these effects were only transient. RhoE induction inhibited cell proliferation and serum-induced S-phase entry. Neither ROCK nor RhoA inhibition accounted for this response. Consistent with its inhibitory effect on cell cycle progression, RhoE expression was induced by cisplatin, a DNA damage-inducing agent. RhoE-expressing cells failed to accumulate cyclin D1 or p21(cip1) protein or to activate E2F-regulated genes in response to serum, although ERK, PI3-K/Akt, FAK, Rac, and cyclin D1 transcription was activated normally. The expression of proteins that bypass the retinoblastoma (pRb) family cell cycle checkpoint, including human papillomavirus E7, adenovirus E1A, and cyclin E, rescued cell cycle progression in RhoE-expressing cells. RhoE also inhibited Ras- and Raf-induced fibroblast transformation. These results indicate that RhoE inhibits cell cycle progression upstream of the pRb checkpoint.