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2.
Exp Hematol ; 122: 41-54, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-37001723

RESUMO

The regenerative potential of human hematopoietic stem cells (HSCs) is functionally defined by their ability to provide life-long blood cell production and to repopulate myeloablated allogeneic transplant recipients. The expansion of HSC numbers is dependent not only on HSC divisions but also on a coordinated adaptation of HSCs to metabolic stress. These variables are especially critical during the ex vivo culture of HSCs with cytokine combinations, which frequently results in HSC exhaustion. We have previously reported that human CD34+ hematopoietic stem and progenitor cells (HSPCs) can be efficiently reprogrammed ex vivo and that the number of phenotypic HSCs with long-term repopulation capacity is expanded in the presence of a combination of cytokines and an epigenetic modifier. Here, we present evidence that ex vivo HSC reprogramming and maintenance is accompanied by increased transcripts of genes regulating metabolic integrity, including SIRT1 and SIRT3.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Humanos , Células-Tronco Hematopoéticas/metabolismo , Citocinas/metabolismo , Antígenos CD34/metabolismo , Sangue Fetal , Células Cultivadas
3.
Blood Rev ; 50: 100853, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34112560

RESUMO

Hematopoietic stem cells (HSCs) have been used for therapeutic purposes for decades in the form of autologous and allogeneic transplantation and are currently emerging as an attractive target for gene therapy. A low stem cell dose is a major barrier to the application of HSC therapy in several situations, primarily umbilical cord blood transplantation and gene modification. Strategies that promote ex vivo expansion of the numbers of functional HSCs could overcome this barrier, hence have been the subject of intense and prolonged research. Several ex vivo expansion strategies have advanced to evaluation clinical trials, which are showing favorable outcomes along with convincing safety signals. Preclinical studies have recently confirmed beneficial incorporation of ex vivo expansion into HSC gene modification protocols. Collectively, ex vivo HSC expansion holds promise for significantly broadening the availability of cord blood units for transplantation, and for optimizing gene therapy protocols to enable their clinical application.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas , Sangue Fetal/fisiologia , Células-Tronco Hematopoéticas/citologia , Humanos
4.
Cytotherapy ; 23(9): 841-851, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34023194

RESUMO

BACKGROUND AIMS: Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative therapy for a wide range of malignant and genetic disorders of the hematopoietic and immune systems. Umbilical cord blood (UCB) is a readily available source of stem cells for allo-HSCT, but the small fixed number of hematopoietic stem and progenitor cells (HSPCs) found in a single unit limits its widespread use in adult recipients. The authors have previously reported that culturing UCB-CD34+ cells in serum-free media supplemented with a combination of cytokines and the histone deacetylase inhibitor valproic acid (VPA) led to expansion of the numbers of functional HSPCs. Such fresh expanded product has been advanced to the clinic and is currently evaluated in an ongoing clinical trial in patients with hematological malignancies undergoing allo-HSCT. Here the authors report on the cryopreservation of this cellular product under current Good Manufacturing Practice (cGMP). METHODS: cGMP VPA-mediated expansion was initiated with CD34+ cells isolated from cryopreserved primary UCB collections, and the functionality after a second cryopreservation step of the expanded product evaluted in vitro and in mouse xenografts. RESULTS: The authors found that the cryopreserved VPA-expanded grafts were characterized by a high degree of viability, retention of HSPC phenotypic subtypes and maintenance of long-term multilineage repopulation capacity in immunocompromised mice. All cellular and functional parameters tested were comparable between the fresh and cryopreserved VPA-expanded cellular products. CONCLUSIONS: The authors' results demonstrate and support the practicality of cryopreservation of VPA-expanded stem cell grafts derived from UCB-CD34+ cells for clinical utilization.


Assuntos
Sangue Fetal , Transplante de Células-Tronco Hematopoéticas , Animais , Antígenos CD34 , Células Cultivadas , Criopreservação , Células-Tronco Hematopoéticas , Xenoenxertos , Humanos , Camundongos
5.
Commun Biol ; 4(1): 517, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941818

RESUMO

Erythropoiesis involves complex interrelated molecular signals influencing cell survival, differentiation, and enucleation. Diseases associated with ineffective erythropoiesis, such as ß-thalassemias, exhibit erythroid expansion and defective enucleation. Clear mechanistic determinants of what make erythropoiesis effective are lacking. We previously demonstrated that exogenous transferrin ameliorates ineffective erythropoiesis in ß-thalassemic mice. In the current work, we utilize transferrin treatment to elucidate a molecular signature of ineffective erythropoiesis in ß-thalassemia. We hypothesize that compensatory mechanisms are required in ß-thalassemic erythropoiesis to prevent apoptosis and enhance enucleation. We identify pleckstrin-2-a STAT5-dependent lipid binding protein downstream of erythropoietin-as an important regulatory node. We demonstrate that partial loss of pleckstrin-2 leads to worsening ineffective erythropoiesis and pleckstrin-2 knockout leads to embryonic lethality in ß-thalassemic mice. In addition, the membrane-associated active form of pleckstrin-2 occurs at an earlier stage during ß-thalassemic erythropoiesis. Furthermore, membrane-associated activated pleckstrin-2 decreases cofilin mitochondrial localization in ß-thalassemic erythroblasts and pleckstrin-2 knockdown in vitro induces cofilin-mediated apoptosis in ß-thalassemic erythroblasts. Lastly, pleckstrin-2 enhances enucleation by interacting with and activating RacGTPases in ß-thalassemic erythroblasts. This data elucidates the important compensatory role of pleckstrin-2 in ß-thalassemia and provides support for the development of targeted therapeutics in diseases of ineffective erythropoiesis.


Assuntos
Apoptose , Núcleo Celular/patologia , Eritroblastos/patologia , Eritropoese , Proteínas de Membrana/fisiologia , Talassemia beta/patologia , Animais , Núcleo Celular/metabolismo , Eritroblastos/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Talassemia beta/etiologia , Talassemia beta/metabolismo
6.
Methods Mol Biol ; 2185: 267-280, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33165854

RESUMO

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic stem cell transplantation but lack a matched donor. However, the limited number of HSCs within each UCB unit remains a major challenge for their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of human HSCs in ex vivo cultures initiated with CD34+ cells isolated from UCBs can overcome this limitation. The method described here utilizes a deacetylase inhibitor, valproic acid (VPA), to rapidly expand to a high degree the numbers of functional HSCs and committed progenitors (HPCs). The expanded HSCs are capable of establishing both short-term and long-term multilineage hematopoietic reconstitution. This highly reproducible and simple protocol can be also applied to expansion of both HSCs and HPCs from different sources including the bone marrow and peripheral blood.


Assuntos
Células-Tronco Adultas/metabolismo , Técnicas de Cultura de Células , Células-Tronco Hematopoéticas/metabolismo , Ácido Valproico/farmacologia , Células-Tronco Adultas/citologia , Células Cultivadas , Células-Tronco Hematopoéticas/citologia , Humanos
7.
Stem Cells Transl Med ; 9(4): 531-542, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31950644

RESUMO

Attempts to expand ex vivo the numbers of human hematopoietic stem cells (HSCs) without compromising their marrow repopulating capacity and their ability to establish multilineage hematopoiesis has been the subject of intense investigation. Although most such efforts have focused on cord blood HSCs, few have been applied to adult HSCs, a more clinically relevant HSC source for gene modification. To date, the strategies that have been used to expand adult HSCs have resulted in modest effects or HSCs with lineage bias and a limited ability to generate T cells in vivo. We previously reported that culturing umbilical cord blood CD34+ cells in serum-free media supplemented with valproic acid (VPA), a histone deacetylase inhibitor, and a combination of cytokines led to the expansion of the numbers of fully functional HSCs. In the present study, we used this same approach to expand the numbers of adult human CD34+ cells isolated from mobilized peripheral blood and bone marrow. This approach resulted in a significant increase in the numbers of phenotypically defined HSCs (CD34+CD45RA-CD90+D49f+). Cells incubated with VPA also exhibited increased aldehyde dehydrogenase activity and decreased mitochondrial membrane potential, each functional markers of HSCs. Grafts harvested from VPA-treated cultures were able to engraft in immune-deficient mice and, importantly, to generate cellular progeny belonging to each hematopoietic lineage in similar proportion to that observed with unmanipulated CD34+ cells. These data support the utility of VPA-mediated ex vivo HSC expansion for gene modification of adult HSCs.


Assuntos
Células-Tronco Adultas/citologia , Células-Tronco Hematopoéticas/citologia , Inibidores de Histona Desacetilases/farmacologia , Adulto , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Contagem de Células , Linhagem da Célula/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Feminino , Sangue Fetal/citologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Fenótipo , Ácido Valproico/farmacologia
8.
Front Cell Dev Biol ; 8: 592348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33384995

RESUMO

Ex vivo expansion strategies of human hematopoietic stem cell (HSC) grafts with suboptimal stem cell dose have emerged as promising strategies for improving outcomes of HSC transplantation in patients with hematological malignancies. While exposure of HSCs to ex vivo cultures expands the number of phenotypically identifiable HSCs, it frequently alters the transcriptomic and metabolic profiles, therefore, compromising their long-term (LT) hematopoietic reconstitution capacity. Within the heterogeneous pool of expanded HSCs, the precise phenotypic, transcriptomic and metabolic profile and thus, the identity of HSCs that confer LT repopulation potential remains poorly described. Utilizing valproic acid (VPA) in ex vivo cultures of umbilical cord blood (UCB)-CD34+ cells, we demonstrate that expanded HSCs phenotypically marked by expression of the stem cell markers CD34, CD90 and EPCR (CD201) are highly enriched for LT-HSCs. Furthermore, we report that low mitochondrial membrane potential, and, hence, mitochondrial activity distinguishes LT-HSCs within the expanded pool of phenotypically defined HSCs. Remarkably, such reduced mitochondrial activity is restricted to cells with the highest expression levels of CD34, CD90 and EPCR phenotypic markers. Together, our findings reveal that high expression of CD34, CD90 and EPCR in conjunction with low mitochondrial activity is critical for identification of functional LT-HSCs generated within ex vivo expansion cultures.

9.
Ann N Y Acad Sci ; 1466(1): 39-50, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31199002

RESUMO

Understanding mechanisms that determine the behavior of human hematopoietic stem cells (HSCs) is essential for developing novel strategies to expand ex vivo the number of fully functional HSCs. In this review, we focus on the complex interplay between intrinsic mechanisms regulated by transcriptional and mitochondrial networks and extrinsic signals imposed by the bone marrow microenvironment, which in concert regulate the balance between HSC self-renewal and differentiation. Such integrated signaling mechanisms that dictate the fate of HSCs in vivo must be recapitulated ex vivo to achieve successful expansion of clinically relevant HSCs. We also highlight some of the most recent ex vivo HSC expansion strategies that have currently entered clinical development. Finally, based on the evidence reviewed here and lessons learned from ex vivo HSC expansion, we raise some critical questions regarding HSC fate and the cellular plasticity of hematopoietic cells that challenge the unidirectional model of human hematopoiesis.


Assuntos
Proliferação de Células , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Animais , Medula Óssea/fisiologia , Diferenciação Celular , Células Cultivadas , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/tendências , Humanos , Modelos Biológicos , Transdução de Sinais/fisiologia
10.
J Vis Exp ; (146)2019 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-31033947

RESUMO

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.


Assuntos
Antígenos CD34/metabolismo , Técnicas de Cultura de Células/métodos , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Cordão Umbilical/citologia , Ácido Valproico/farmacologia , Adulto , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Antígenos Thy-1/metabolismo
11.
Stem Cells Int ; 2019: 4067162, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30881461

RESUMO

Quiescent and self-renewing hematopoietic stem cells (HSCs) rely on glycolysis rather than on mitochondrial oxidative phosphorylation (OxPHOS) for energy production. HSC reliance on glycolysis is considered an adaptation to the hypoxic environment of the bone marrow (BM) and reflects the low energetic demands of HSCs. Metabolic rewiring from glycolysis to mitochondrial-based energy generation accompanies HSC differentiation and lineage commitment. Recent evidence, however, highlights that alterations in mitochondrial metabolism and activity are not simply passive consequences but active drivers of HSC fate decisions. Modulation of mitochondrial activity and metabolism is therefore critical for maintaining the self-renewal potential of primitive HSCs and might be beneficial for ex vivo expansion of transplantable HSCs. In this review, we emphasize recent advances in the emerging role of mitochondria in hematopoiesis, cellular reprograming, and HSC fate decisions.

12.
Blood Adv ; 2(20): 2766-2779, 2018 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-30348672

RESUMO

The limited number of hematopoietic stem cells (HSCs) in umbilical cord blood (UCB) units restricts their use for stem cell transplantation. Ex vivo treatment of UCB-CD34+ cells with valproic acid (VPA) increases the number of transplantable HSCs. In this study, we demonstrate that HSC expansion is not merely a result of proliferation of the existing stem cells but, rather, a result of a rapid reprogramming of CD34+CD90- cells into CD34+CD90+ cells, which is accompanied by limited numbers of cell divisions. Beyond this phenotypic switch, the treated cells acquire and retain a transcriptomic and mitochondrial profile, reminiscent of primary HSCs. Single and bulk RNA-seq revealed a signature highly enriched for transcripts characteristic of primary HSCs. The acquisition of this HSC signature is linked to mitochondrial remodeling accompanied by a reduced activity and enhanced glycolytic potential. These events act in concert with a modest upregulation of p53 activity to limit the levels of reactive oxygen species (ROS). Inhibition of either glycolysis or p53 activity impairs HSC expansion. This study indicates that a complex interplay of events is required for effective ex vivo expansion of UCB-HSCs.


Assuntos
Reprogramação Celular/genética , Células-Tronco Hematopoéticas/metabolismo , Mitocôndrias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos
14.
Genes Cancer ; 5(1-2): 15-21, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24955214

RESUMO

Cancer cells have elevated levels of reactive oxygen species (ROS), which are generated in majority by the mitochondria. In the mitochondrial matrix, the manganese dismutase SOD2 acts as a major anti-oxidant enzyme. The deacetylase SIRT3 regulates the activity of SOD2. Recently, SIRT3 was reported to be decreased in 87% of breast cancers, resulting therefore in a decrease in the activity of SOD2 and an elevation in ROS. In addition to SIRT3, we recently reported that SOD2 itself is down-regulated in breast cancer cell lines upon activation of oncogenes, such as Ras. Since in absence of SOD2, superoxide levels are elevated and may cause irreversible damage, mechanisms must exist to retain superoxide below a critical threshold and maintain viability of cancer cells. The copper/zinc dismutase SOD1 localizes in the cytoplasm, the inter-membrane space of the mitochondria and the nucleus. Emerging evidences from several groups now indicate that SOD1 is overexpressed in cancers and that the activity of SOD1 may be essential to maintain cellular ROS under this critical threshold. This review summarizes the studies reporting important roles of SOD1 in cancer and addresses the potential cross-talk between the overexpression of SOD1 and the regulation of the mitochondrial unfolded protein response (UPR(mt)). While mutations in SOD1 is the cause of 20% of cases of familial amyotrophic lateral sclerosis (fALS), a devastating neurodegenerative disease, these new studies expand the role of SOD1 to cancer.

15.
J Biol Chem ; 289(9): 5412-6, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24448804

RESUMO

Cancer cells are characterized by elevated levels of reactive oxygen species, which are produced mainly by the mitochondria. The dismutase SOD2 localizes in the matrix and is a major antioxidant. The activity of SOD2 is regulated by the deacetylase SIRT3. Recent studies indicated that SIRT3 is decreased in 87% of breast cancers, implying that the activity of SOD2 is compromised. The resulting elevation in reactive oxygen species was shown to be essential for the metabolic reprograming toward glycolysis. Here, we show that SOD2 itself is down-regulated in breast cancer cell lines. Further, activation of oncogenes, such as Ras, promotes the rapid down-regulation of SOD2. Because in the absence of SOD2, superoxide levels are elevated in the matrix, we reasoned that mechanisms must exist to retain low levels of superoxide in other cellular compartments especially in the intermembrane space of the mitochondrial to avoid irreversible damage. The dismutase SOD1 also acts as an antioxidant, but it localizes to the cytoplasm and the intermembrane space of the mitochondria. We report here that loss of SOD2 correlates with the overexpression of SOD1. Further, we show that mitochondrial SOD1 is the main dismutase activity in breast cancer cells but not in non-transformed cells. In addition, we show that the SOD1 inhibitor LCS-1 leads to a drastic fragmentation and swelling of the matrix, suggesting that in the absence of SOD2, SOD1 is required to maintain the integrity of the organelle. We propose that by analogy to the cadherin switch during epithelial-mesenchymal transition, cancer cells also undergo a SOD switch during transformation.


Assuntos
Neoplasias da Mama/enzimologia , Transição Epitelial-Mesenquimal , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Superóxido Dismutase/biossíntese , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Sirtuína 3/genética , Sirtuína 3/metabolismo , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , Superóxido Dismutase-1 , Superóxidos/metabolismo
16.
Mol Cell Biol ; 34(4): 699-710, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24324009

RESUMO

The mitochondria of cancer cells are characterized by elevated oxidative stress caused by reactive oxygen species (ROS). Such an elevation in ROS levels contributes to mitochondrial reprogramming and malignant transformation. However, high levels of ROS can cause irreversible damage to proteins, leading to their misfolding, mitochondrial stress, and ultimately cell death. Therefore, mechanisms to overcome mitochondrial stress are needed. The unfolded protein response (UPR) triggered by accumulation of misfolded proteins in the mitochondria (UPR(mt)) has been reported recently. So far, the UPR(mt) has been reported to involve the activation of CHOP and estrogen receptor alpha (ERα). The current study describes a novel role of the mitochondrial deacetylase SirT3 in the UPR(mt). Our data reveal that SirT3 acts to orchestrate two pathways, the antioxidant machinery and mitophagy. Inhibition of SirT3 in cells undergoing proteotoxic stress severely impairs the mitochondrial network and results in cellular death. These observations suggest that SirT3 acts to sort moderately stressed from irreversibly damaged organelles. Since SirT3 is reported to act as a tumor suppressor during transformation, our findings reveal a dual role of SirT3. This novel role of SirT3 in established tumors represents an essential mechanism of adaptation of cancer cells to proteotoxic and mitochondrial stress.


Assuntos
Mitocôndrias/metabolismo , Sirtuína 3/metabolismo , Resposta a Proteínas não Dobradas/genética , Acetilação , Morte Celular/genética , Morte Celular/fisiologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Humanos , Mitocôndrias/genética , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Ligação Proteica/genética , Espécies Reativas de Oxigênio/metabolismo , Resposta a Proteínas não Dobradas/fisiologia
17.
Sci Transl Med ; 5(169): 169ra12, 2013 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-23345610

RESUMO

Metastasis is the major cause of cancer mortality. A more thorough understanding of the mechanisms driving this complex multistep process will aid in the identification and characterization of therapeutically targetable genetic drivers of disease progression. We demonstrate that KLF6-SV1, an oncogenic splice variant of the KLF6 tumor suppressor gene, is associated with increased metastatic potential and poor survival in a cohort of 671 lymph node-negative breast cancer patients. KLF6-SV1 overexpression in mammary epithelial cell lines resulted in an epithelial-to-mesenchymal-like transition and drove aggressive multiorgan metastatic disease in multiple in vivo models. Additionally, KLF6-SV1 loss-of-function studies demonstrated reversion to an epithelial and less invasive phenotype. Combined, these findings implicate KLF6-SV1 as a key driver of breast cancer metastasis that distinguishes between indolent and lethal early-stage disease and provides a potential therapeutic target for invasive breast cancer.


Assuntos
Processamento Alternativo/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Fatores de Transcrição Kruppel-Like/genética , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Animais , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Pessoa de Meia-Idade , Análise Multivariada , Invasividade Neoplásica , Metástase Neoplásica/genética , Proteínas Nucleares , Fenótipo , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sobrevida , Proteína 1 Relacionada a Twist , Ensaios Antitumorais Modelo de Xenoenxerto
18.
J Cell Sci ; 124(Pt 9): 1396-402, 2011 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-21486948

RESUMO

Unfolded protein responses (UPRs) of the endoplasmic reticulum and mitochondrial matrix have been described. Here, we show that the accumulation of proteins in the inter-membrane space (IMS) of mitochondria in the breast cancer cell line MCF-7 activates a distinct UPR. Upon IMS stress, overproduction of reactive oxygen species (ROS) and phosphorylation of AKT triggers estrogen receptor (ER) activity, which further upregulates the transcription of the mitochondrial regulator NRF1 and the IMS protease OMI (officially known as HTRA2). Moreover, we demonstrate that the IMS stress-induced UPR culminates in increased proteasome activity. Given our previous report on a proteasome- and OMI-dependent checkpoint that limits the import of IMS proteins, the findings presented in this study suggest that this newly discovered UPR acts as a cytoprotective response to overcome IMS stress.


Assuntos
Mitocôndrias/metabolismo , Receptores de Estrogênio/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Western Blotting , Linhagem Celular Tumoral , Serina Peptidase 2 de Requerimento de Alta Temperatura A , Humanos , Potencial da Membrana Mitocondrial/genética , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fator 1 Nuclear Respiratório/genética , Fator 1 Nuclear Respiratório/metabolismo , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Fosforilação/genética , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de Estrogênio/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Resposta a Proteínas não Dobradas/genética
19.
Clin Cancer Res ; 17(8): 2292-300, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21292820

RESUMO

PURPOSE: Fulvestrant is known to promote the degradation of the estrogen receptor (ER) in the nucleus. However, fulvestrant also promotes the aggregation of the newly synthesized ER in the cytoplasm. Accumulation of protein aggregates leads to cell death but this effect is limited as a result of their elimination by the proteasome. We tested whether combining fulvestrant with the proteasome inhibitor, bortezomib, could enhance the accumulation of ER aggregates and cause apoptotic cell death. EXPERIMENTAL DESIGN: The rate of aggregation of the ER was monitored in ER(+) breast cancer cells lines, T47D, ZR-75.1, BT474, MDA-MB-361, MCF-7, fulvestrant resistance MCF-7, and tamoxifen-resistant T47D-cyclin D1 cells. Activation of the unfolded protein response, apoptosis, and metabolic rate were also monitored in these cell lines following treatment with fulvestrant, bortezomib, or bortezomib in combination with fulvestrant. RESULTS: We found that bortezomib enhances the fulvestrant-mediated aggregation of the ER in the cytoplasm without blocking the degradation of the ER in the nucleus. Further, these aggregates activate a sustained unfolded protein response leading to apoptotic cell death. Further, we show that the combination induced tumor regression in a breast cancer mouse model of tamoxifen resistance. CONCLUSIONS: Adding bortezomib to fulvestrant enhances its efficacy by taking advantage of the unique ability of fulvestrant to promote cytoplasmic aggregates of the ER. As this effect of fulvestrant is independent of the transcriptional activity of the ER, these results suggest that this novel combination may be effective in breast cancers that are ER(+) but estrogen independent.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Receptores de Estrogênio/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Antineoplásicos Hormonais/farmacologia , Western Blotting , Ácidos Borônicos/administração & dosagem , Ácidos Borônicos/farmacologia , Bortezomib , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Sinergismo Farmacológico , Estradiol/administração & dosagem , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Imunofluorescência , Fulvestranto , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pirazinas/administração & dosagem , Pirazinas/farmacologia , Receptores de Estrogênio/genética , Tamoxifeno/farmacologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Apoptosis ; 13(4): 588-99, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18299995

RESUMO

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored.


Assuntos
Sobrevivência Celular/fisiologia , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Oligopeptídeos/farmacologia , Estresse Oxidativo/fisiologia , Inibidores de Proteassoma , Acetilcisteína/farmacologia , Animais , Caspases/metabolismo , Morte Celular/fisiologia , Células Cultivadas , Hipocampo/citologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Mitocôndrias/fisiologia , Modelos Biológicos , Neurônios/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteína bcl-X/biossíntese , Proteína bcl-X/farmacologia
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