RESUMEN
HMGB3 protein belongs to the group of HMGB proteins from the superfamily of nuclear proteins with high electrophoretic mobility. HMGB proteins play an active part in almost all cellular processes associated with DNA-repair, replication, recombination, and transcription-and, additionally, can act as cytokines during infectious processes, inflammatory responses, and injuries. Although the structure and functions of HMGB1 and HMGB2 proteins have been intensively studied for decades, very little attention has been paid to HMGB3 until recently. In this review, we summarize the currently available data on the molecular structure, post-translational modifications, and biological functions of HMGB3, as well as the possible role of the ubiquitin-proteasome system-dependent HMGB3 degradation in tumor development.
Asunto(s)
Proteína HMGB3 , Procesamiento Proteico-Postraduccional , Humanos , Proteína HMGB3/metabolismo , Proteína HMGB3/química , Proteína HMGB3/genética , Animales , Neoplasias/metabolismo , Proteolisis , Complejo de la Endopetidasa Proteasomal/metabolismoRESUMEN
High-Mobility Group (HMG) chromosomal proteins are the most numerous nuclear non-histone proteins. HMGB domain proteins are the most abundant and well-studied HMG proteins. They are involved in variety of biological processes. HMGB1 and HMGB2 were the first members of HMGB-family to be discovered and are found in all studied eukaryotes. Despite the high degree of homology, HMGB1 and HMGB2 proteins differ from each other both in structure and functions. In contrast to HMGB2, there is a large pool of works devoted to the HMGB1 protein whose structure-function properties have been described in detail in our previous review in 2020. In this review, we attempted to bring together diverse data about the structure and functions of the HMGB2 protein. The review also describes post-translational modifications of the HMGB2 protein and its role in the development of a number of diseases. Particular attention is paid to its interaction with various targets, including DNA and protein partners. The influence of the level of HMGB2 expression on various processes associated with cell differentiation and aging and its ability to mediate the differentiation of embryonic and adult stem cells are also discussed.
Asunto(s)
Proteína HMGB1 , Proteína HMGB2 , Proteína HMGB2/genética , Proteína HMGB2/metabolismo , Proteína HMGB1/metabolismo , Proteínas HMGB/metabolismo , Factores de Transcripción , ADN/metabolismo , Proteínas Nucleares , Proteínas del Grupo de Alta MovilidadRESUMEN
Non-histone nuclear proteins HMGB1 and HMGB2 (High Mobility Group) are involved in many biological processes, such as replication, transcription, and repair. The HMGB1 and HMGB2 proteins consist of a short N-terminal region, two DNA-binding domains, A and B, and a C-terminal sequence of glutamic and aspartic acids. In this work, the structural organization of calf thymus HMGB1 and HMGB2 proteins and their complexes with DNA were studied using UV circular dichroism (CD) spectroscopy. Post-translational modifications (PTM) of HMGB1 and HMGB2 proteins were determined with MALDI mass spectrometry. We have shown that despite the similar primary structures of the HMGB1 and HMGB2 proteins, their post-translational modifications (PTMs) demonstrate quite different patterns. The HMGB1 PTMs are located predominantly in the DNA-binding A-domain and linker region connecting the A and B domains. On the contrary, HMGB2 PTMs are found mostly in the B-domain and within the linker region. It was also shown that, despite the high degree of homology between HMGB1 and HMGB2, the secondary structure of these proteins is also slightly different. We believe that the revealed structural properties might determine the difference in the functioning of the HMGB1 and HMGB2 as well as their protein partners.
Asunto(s)
Proteína HMGB1 , Proteína HMGB2 , ADN/química , ADN/metabolismo , Proteínas del Grupo de Alta Movilidad , Proteína HMGB1/química , Proteína HMGB1/metabolismo , Proteína HMGB2/química , Proteína HMGB2/metabolismo , Factores de Transcripción , Unión Proteica , Animales , BovinosRESUMEN
The monoamine neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has important functions both in the neural system and during embryonic development in mammals. In this study, we set out to investigate whether and how endogenous serotonin affects reprogramming to pluripotency. As serotonin is synthesized from tryptophan by the rate limiting enzymes tryptophan hydroxylase-1 and -2 (TPH1 and TPH2), we have assessed the reprogramming of TPH1- and/or TPH2-deficient mouse embryonic fibroblasts (MEFs) to induced pluripotent stem cells (iPSCs). The reprogramming of the double mutant MEFs showed a dramatic increase in the efficiency of iPSC generation. In contrast, ectopic expression of TPH2 alone or in conjunction with TPH1 reverted the rate of reprogramming of the double mutant MEFs to the wild-type level and besides, TPH2 overexpression significantly suppressed reprogramming of wild-type MEFs. Our data thus suggest a negative role of serotonin biosynthesis in the reprogramming of somatic cells to a pluripotent state.
Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes , Serotonina , Triptófano Hidroxilasa , Animales , Ratones , Fibroblastos/metabolismo , Serotonina/biosíntesis , Triptófano/metabolismo , Triptófano Hidroxilasa/metabolismoRESUMEN
The transcription factor Oct4 can rightfully be considered a pivotal element in maintaining pluripotency. In addition, its ability to function as a pioneer factor enables the reprogramming of somatic cells back into a pluripotent state. To better understand the regulation of the Oct4-encoding gene (Pou5f1), the main genetic elements that regulate its expression in different states of pluripotency ought to be identified. While some elements have been well characterized for their ability to drive Pou5f1 expression, others have yet to be determined. In this work, we show that translocation of the Pou5f1 gene fragment purported to span all essential cis-elements, including the well-known distal and proximal enhancers (DE and PE), into the Rosa26 locus impairs the self-renewal of mouse embryonic stem cells (ESCs) in the naïve pluripotency state, as well as their further advancement through the formative and primed pluripotency states, inducing overall differentiation failure. These results suggest that regulatory elements located outside the previously determined Pou5f1 boundaries are critical for the proper spatiotemporal regulation of this gene during development, indicating the need for their better characterization.
Asunto(s)
Células Madre Embrionarias , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Ratones , Secuencias Reguladoras de Ácidos Nucleicos/genética , Diferenciación Celular/genética , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
Oct4, a class V POU-domain protein that is encoded by the Pou5f1 gene, is thought to be a key transcription factor in the early development of mammals. This transcription factor plays indispensable roles in pluripotent stem cells as well as in the acquisition of pluripotency during somatic cell reprogramming. Oct4 has also been shown to play a role as a pioneer transcription factor during zygotic genome activation (ZGA) from zebrafish to human. However, during the past decade, several studies have brought these conclusions into question. It was clearly shown that the first steps in mouse development are not affected by the loss of Oct4. Subsequently, the role of Oct4 as a genome activator was brought into doubt. It was also found that the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) could proceed without Oct4. In this review, we summarize recent findings, reassess the role of Oct4 in reprogramming and ZGA, and point to structural features that may underlie this role. We speculate that pluripotent stem cells resemble neural stem cells more closely than previously thought. Oct4 orthologs within the POUV class hold key roles in genome activation during early development of species with late ZGA. However, in Placentalia, eutherian-specific proteins such as Dux overtake Oct4 in ZGA and endow them with the formation of an evolutionary new tissue-the placenta.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/citología , Células-Madre Neurales/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Activación Transcripcional/fisiología , Animales , Reprogramación Celular , Dimerización , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Placenta/fisiología , Embarazo , Pez CebraRESUMEN
A novel approach in gene therapy was introduced 20 years ago since artificial non-integrative chromosome-based vectors containing gene loci size inserts were engineered. To date, different human artificial chromosomes (HAC) were generated with the use of de novo construction or "top-down" engineering approaches. The HAC-based therapeutic approach includes ex vivo gene transferring and correction of pluripotent stem cells (PSCs) or highly proliferative modified stem cells. The current progress in the technology of induced PSCs, integrating with the HAC technology, resulted in a novel platform of stem cell-based tissue replacement therapy for the treatment of genetic disease. Nowadays, the sophisticated and laborious HAC technology has significantly improved and is now closer to clinical studies. In here, we reviewed the achievements in the technology of de novo synthesized HACs for a chromosome transfer for developing gene therapy tissue replacement models of monogenic human diseases.
Asunto(s)
Cromosomas Artificiales Humanos/genética , Terapia Genética , Células Madre Pluripotentes Inducidas/trasplante , Trasplante de Células Madre , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , HumanosRESUMEN
Tools developed in the fields of genome engineering, precise gene regulation, and synthetic gene networks have an increasing number of applications. When shared with the scientific community, these tools can be used to further unlock the potential of precision medicine and tissue engineering. A large number of different genetic elements, as well as modifications, have been used to create many different systems and to validate some technical concepts. New studies have tended to optimize or improve existing elements or approaches to create complex synthetic systems, especially those based on the relatively new CRISPR technology. In order to maximize the output of newly developed approaches and to move from proof-of-principle experiments to applications in regenerative medicine, it is important to navigate efficiently through the vast number of genetic elements to choose those most suitable for specific needs. In this review, we have collected information regarding the main genetic elements and their modifications, which can be useful in different synthetic systems with an emphasis of those based on CRISPR technology. We have indicated the most suitable elements and approaches to choose or combine in planning experiments, while providing their deeper understanding, and have also stated some pitfalls that should be avoided.
Asunto(s)
Sistemas CRISPR-Cas , Ingeniería Genética , Sistemas CRISPR-Cas/genética , Genoma , Ingeniería de Tejidos , Redes Reguladoras de Genes , Edición GénicaRESUMEN
Essential changes in cell metabolism and redox signaling occur during the reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). In this paper, using genetic and pharmacological approaches, we have investigated the role of electron transport chain (ETC) complex-I (CI) of mitochondria in the process of cell reprogramming to pluripotency. Knockdown of NADH-ubiquinone oxidoreductase core subunits S1 (Ndufs1) or subunit B10 (Ndufb10) of the CI or inhibition of this complex with rotenone during mouse embryonic fibroblast (MEF) reprogramming resulted in a significantly decreased number of induced pluripotent stem cells (iPSCs). We have found that mitochondria and ROS levels due course of the reprogramming tightly correlate with each other, both reaching peak by day 3 and significantly declining by day 10 of the process. The transient augmentation of mitochondrial reactive oxygen species (ROS) could be attenuated by antioxidant treatment, which ameliorated overall reprogramming. However, ROS scavenging after day 3 or during the entire course of reprogramming was suppressive for iPSC formation. The ROS scavenging within the CI-deficient iPSC-precursors did not improve, but further suppressed the reprogramming. Our data therefore point to distinct modes of mitochondrial ROS action during the early versus mid and late stages of reprogramming. The data further substantiate the paradigm that balanced levels of oxidative phosphorylation have to be maintained on the route to pluripotency.
Asunto(s)
Complejo I de Transporte de Electrón , Células Madre Pluripotentes Inducidas , Animales , Antioxidantes/metabolismo , Reprogramación Celular/genética , Complejo I de Transporte de Electrón/genética , Complejo I de Transporte de Electrón/metabolismo , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rotenona/metabolismo , Rotenona/farmacologíaRESUMEN
The gene therapy approach aiming at curing various human diseases began to develop as a technology from early eighties of the last century. To date the delivery of therapeutic genes are mainly mediated by virus-based, predominantly, non-integrated virus vectors. These gene delivery approaches have several fundamental limitations on the way of efficient deployment in clinical gene therapy. A totally different approach was suggested about 20 years ago when episomal non-integrative artificial chromosome-based vectors featuring large size inserts (even native gene loci) advanced to the stage. Since then numerous human artificial chromosome (HAC) vectors were developed by both de novo synthesis and top-down engineering technology. This approach so far is limited to ex vivo gene transfer and correction within highly proliferative or reversibly immortalized precursor stem cells or pluripotent stem cells. Recent breakthrough in generation of induced pluripotent stem cells and embryonic stem cell manipulation give the additional pivotal stimuli to integrate it with the HAC technology and to develop thereby novel approaches to replacement therapies of human genetic diseases. The HAC technology is complex and time consuming while nowadays it has significantly advanced and become notably closer to medical applications. In this review we discuss current advancements in the HAC technology, in particular, in terms of improvement of chromosome transfer method and achievements in developing mouse-based gene therapy tissue replacement models for several monogenic human diseases. The main progress has been done in elaboration of top-down type HAC technology in modeling and preclinical studies of gene therapy treatment for Duchenne muscular dystrophy (DMD) disease.
Asunto(s)
Cromosomas Artificiales Humanos/fisiología , Terapia Genética/métodos , Células Madre Pluripotentes/trasplante , Trasplante de Células Madre/métodos , Células Madre Embrionarias/fisiología , Técnicas de Transferencia de Gen , Terapia Genética/efectos adversos , Terapia Genética/ética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Células Madre Pluripotentes/metabolismo , Trasplante de Células Madre/efectos adversos , Trasplante de Células Madre/éticaRESUMEN
The transcription factor Oct4 plays a key regulatory role in the induction and maintenance of cellular pluripotency. In this article, we show that ubiquitous and multifunctional poly(C) DNA/RNA-binding protein hnRNP-K occupies Oct4 (Pou5f1) enhancers in embryonic stem cells (ESCs) but is dispensable for the initiation, maintenance, and downregulation of Oct4 gene expression. Nevertheless, hnRNP-K has an essential cell-autonomous function in ESCs to maintain their proliferation and viability. To better understand mechanisms of hnRNP-K action in ESCs, we have performed ChIP-seq analysis of genome-wide binding of hnRNP-K and identified several thousands of hnRNP-K target sites that are frequently co-occupied by pluripotency-related and common factors (Oct4, TATA-box binding protein, Sox2, Nanog, Otx2, etc.), as well as active histone marks. Furthermore, hnRNP-K localizes exclusively within open chromatin, implying its role in the onset and/or maintenance of this chromatin state. Stem Cells 2019;37:1018-1029.
Asunto(s)
Proliferación Celular , Cromatina/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Supervivencia Celular , Cromatina/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ratones , Factores de Transcripción/genéticaRESUMEN
The functioning of DNA in the cell nucleus is ensured by a multitude of proteins, whose interactions with DNA as well as with other proteins lead to the formation of a complicated, organized, and quite dynamic system known as chromatin. This review is devoted to the description of properties and structure of the progenitors of the most abundant non-histone protein of the HMGB family-the HmgB1 protein. The proteins of the HMGB family are also known as "architectural factors" of chromatin, which play an important role in gene expression, transcription, DNA replication, and repair. However, as soon as HmgB1 goes outside the nucleus, it acquires completely different functions, post-translational modifications, and change of its redox state. Despite a lot of evidence of the functional activity of HmgB1, there are still many issues to be solved related to the mechanisms of the influence of HmgB1 on the development and treatment of different diseases-from oncological and cardiovascular diseases to pathologies during pregnancy and childbirth. Here, we describe molecular structure of the HmgB1 protein and discuss general mechanisms of its interactions with other proteins and DNA in cell.
Asunto(s)
Enfermedades Cardiovasculares/metabolismo , ADN/metabolismo , Proteína HMGB1/metabolismo , Neoplasias/metabolismo , Animales , Cromatina/metabolismo , Reparación del ADN , Replicación del ADN , Regulación de la Expresión Génica , Humanos , Procesamiento Proteico-Postraduccional , Transcripción GenéticaRESUMEN
The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways in eukaryotic cells. Abnormal functioning of this system has been observed in cancer and neurological diseases. The 20S proteasomes, essential components of the UPS, are present not only within the cells but also in the extracellular space, and their concentration in blood plasma has been found to be elevated and dependent upon the disease state, being of prognostic significance in patients suffering from cancer, liver diseases, and autoimmune diseases. However, functions of extracellular proteasomes and mechanisms of their release by cells remain largely unknown. The main mechanism of proteasome activity regulation is provided by modulation of their composition and post-translational modifications (PTMs). Moreover, diverse PTMs of proteins are known to participate in the loading of specific elements into extracellular vesicles. Since previous studies have revealed that the transport of extracellular proteasomes may occur via extracellular vesicles, we have set out to explore the PTMs of extracellular proteasomes in comparison to cellular counterparts. In this work, cellular and extracellular proteasomes were affinity purified and separated by SDS-PAGE for subsequent trypsinization and matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. In total, we could identify 64 and 55 PTM sites in extracellular and cellular proteasomes, respectively, including phosphorylation, ubiquitination, acetylation, and succinylation. We observed novel sites of acetylation at K238 and K192 of the proteasome subunits ß2 and ß3, respectively, that are specific for extracellular proteasomes. Moreover, cellular proteasomes show specific acetylation at K227 of α2 and ubiquitination at K201 of ß3. Interestingly, succinylation of ß6 at the residue K228 seems not to be present exclusively in extracellular proteasomes, whereas both extracellular and cellular proteasomes may also be acetylated at this site. The same situation takes place at K201 of the ß3 subunit where ubiquitination is seemingly specific for cellular proteasomes. Moreover, crosstalk between acetylation, ubiquitination, and succinylation has been observed in the subunit α3 of both proteasome populations. These data will serve as a basis for further studies, aimed at dissection of the roles of extracellular proteasome-specific PTMs in terms of the function of these proteasomes and mechanism of their transport into extracellular space.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Secuencia de Aminoácidos , Humanos , Células K562 , Péptidos/análisis , Péptidos/química , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
The ubiquitin proteasome system is involved in the regulation of most basic intracellular processes, and deregulation of this system can results in certain kinds of human diseases. Proteolytic core this system, the 20S proteasome, has been found in physiological fluids of both healthy humans and patients suffering from a variety of inflammatory, autoimmune, and neoplastic diseases. The concentration of these extracellular proteasomes has been found to correlate with the diseased state, being of a prognostic significance. The transport mechanisms and functions of these proteasomes, however, are largely unclear. Previous studies revealed that the transport of extracellular proteasomes may occur via microvesicles and exosomes, which led to the hypothesis that extracellular proteasomes are implicated in cell-to-cell communication process. Here we show that microvesicles and exosomes, two major known types of intercellular vehicles, contain no detectable proteasomes. Moreover, neither affinity purified nor naturally released into conditioned medium by donor cells 20S proteasomes could penetrate recipient HeLa cells. Taken together, these results suggest that extracellular proteasomes are unlikely to be involved in the cell-to-cell communication and that their release by cells serve other biological purposes.
Asunto(s)
Comunicación Celular/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Micropartículas Derivadas de Células/metabolismo , Exosomas , Espacio Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Células K562 , Modelos Biológicos , Transporte de Proteínas , Proteínas Recombinantes/metabolismoRESUMEN
During T cell-dependent (TD) germinal center (GC) responses, naïve B cells are instructed to differentiate towards GC B cells (GCBC), high-affinity long-lived plasma cells (LLPC) or memory B cells (Bmem). Alterations in the B cell-fate choice could contribute to immune dysregulation leading to the loss of self-tolerance and the initiation of autoimmune disease. Here we show that mRNA levels of the transcription regulator BOB.1 are increased in the lymph node compartment of patients with rheumatoid arthritis (RA), a prototypical autoimmune disease caused by the loss of immunological tolerance. Investigating to what extent levels of BOB.1 impact B cells during TD immune responses we found that BOB.1 has a crucial role in determining the B cell-fate decision. High BOB.1 levels promote the generation of cells with phenotypic and functional characteristics of Bmem. Mechanistically, overexpression of BOB.1 drives ABF1 and suppresses BCL6, favouring Bmem over LLPC or recycling GCBC. Low levels of BOB.1 are sufficient for LLPC but not for Bmem differentiation. Our findings demonstrate a novel role for BOB.1 in B cells during TD GC responses and suggest that its dysregulation may contribute to the pathogenesis of RA by disturbing the B cell-fate determination.
Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Centro Germinal/inmunología , Centro Germinal/metabolismo , Memoria Inmunológica/genética , Transactivadores/genética , Animales , Biomarcadores , Línea Celular , Expresión Génica , Humanos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ratones , Ratones Noqueados , Células Plasmáticas/inmunología , Células Plasmáticas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Fiebre Reumática/genética , Fiebre Reumática/inmunología , Fiebre Reumática/metabolismo , Fiebre Reumática/patología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Bicuspid aortic valve is the most common congenital heart malformation and the reasons for the aortopathies associated with bicuspid aortic valve remain unclear. NOTCH1 mutations are associated with bicuspid aortic valve and have been found in individuals with various left ventricular outflow tract abnormalities. Notch is a key signaling during cardiac valve formation that promotes the endothelial-to-mesenchymal transition. We address the role of Notch signaling in human aortic endothelial cells from patients with bicuspid aortic valve and aortic aneurysm. Aortic endothelial cells were isolated from tissue fragments of bicuspid aortic valve-associated thoracic aortic aneurysm patients and from healthy donors. Endothelial-to-mesenchymal transition was induced by activation of Notch signaling. Effectiveness of the transition was estimated by loss of endothelial and gain of mesenchymal markers by immunocytochemistry and qPCR. We show that aortic endothelial cells from the patients with aortic aneurysm and bicuspid aortic valve have down regulated Notch signaling and fail to activate Notch-dependent endothelial-to-mesenchymal transition in response to its stimulation by different Notch ligands. Our findings support the idea that bicuspid aortic valve and associated aortic aneurysm is associated with dysregulation of the entire Notch signaling pathway independently on the specific gene mutation.
Asunto(s)
Aneurisma de la Aorta/metabolismo , Válvula Aórtica/anomalías , Endotelio Vascular/metabolismo , Enfermedades de las Válvulas Cardíacas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Adulto , Aneurisma de la Aorta/patología , Válvula Aórtica/metabolismo , Válvula Aórtica/patología , Enfermedad de la Válvula Aórtica Bicúspide , Endotelio Vascular/patología , Femenino , Enfermedades de las Válvulas Cardíacas/patología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
The principal cause of death in cancer involves tumor progression and metastasis. Since only a small proportion of the primary tumor cells, cancer stem cells (CSCs), which are the most aggressive, have the capacity to metastasize and display properties of stem cells, it is imperative to characterize the gene expression of diagnostic markers and to evaluate the drug sensitivity in the CSCs themselves. Here, we have examined the key genes that are involved in the progression of colorectal cancer and are expressed in cancer stem cells. Primary cultures of colorectal cancer cells from a patient's tumors were studied using the flow cytometry and cytological methods. We have evaluated the clinical and stem cell marker expression in these cells, their resistance to 5-fluorouracil and irinotecan, and the ability of cells to form tumors in mice. The data shows the role of stem cell marker Oct4 in the resistance of primary colorectal cancer tumor cells to 5-fluorouracil.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias del Colon/genética , Resistencia a Antineoplásicos/genética , Células Madre Neoplásicas/metabolismo , Animales , Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Camptotecina/análogos & derivados , Camptotecina/farmacología , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Fluorouracilo/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Irinotecán , Ratones , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Oct4 is a widely recognized pluripotency factor as it maintains Embryonic Stem (ES) cells in a pluripotent state, and, in vivo, prevents the inner cell mass (ICM) in murine embryos from differentiating into trophectoderm. However, its function in somatic tissue after this developmental stage is not well characterized. Using a tamoxifen-inducible Cre recombinase and floxed alleles of Oct4, we investigated the effect of depleting Oct4 in mouse embryos between the pre-streak and headfold stages, ~E6.0-E8.0, when Oct4 is found in dynamic patterns throughout the embryonic compartment of the mouse egg cylinder. We found that depletion of Oct4 ~E7.5 resulted in a severe phenotype, comprised of craniorachischisis, random heart tube orientation, failed turning, defective somitogenesis and posterior truncation. Unlike in ES cells, depletion of the pluripotency factors Sox2 and Oct4 after E7.0 does not phenocopy, suggesting that ~E7.5 Oct4 is required within a network that is altered relative to the pluripotency network. Oct4 is not required in extraembryonic tissue for these processes, but is required to maintain cell viability in the embryo and normal proliferation within the primitive streak. Impaired expansion of the primitive streak occurs coincident with Oct4 depletion â¼E7.5 and precedes deficient convergent extension which contributes to several aspects of the phenotype.
Asunto(s)
Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Linaje de la Célula , Proliferación Celular , Desarrollo Embrionario , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Ratones , Defectos del Tubo Neural/etiología , Defectos del Tubo Neural/genética , Defectos del Tubo Neural/patología , Factor 3 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/citología , Línea Primitiva/crecimiento & desarrollo , Línea Primitiva/metabolismo , Factores de Transcripción SOXB1/antagonistas & inhibidores , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoid(tetO)-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoid(tetO)-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors.
Asunto(s)
Cromatina/genética , Cromosomas Artificiales Humanos , Vectores Genéticos/genética , Transgenes , Animales , Células CHO , Línea Celular Tumoral , Cricetinae , Cricetulus , ADN Satélite/genética , Expresión Génica , Silenciador del Gen , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , ARN de Transferencia/genéticaRESUMEN
BOB1, a mammalian lymphocyte-specific transcriptional coactivator of the transcription factors OCT1 and OCT2 (OCT1/2), plays important roles in normal immune responses, autoimmunity, and hematologic malignancies. The issue of a DNA sequence preference change imposed by BOB1 was raised more than two decades ago but remains unresolved. In this paper, using the EMSA-SELEX-Seq approach, we have reassessed the intrinsic ability of BOB1 to modulate the specificity of DNA recognition by OCT1 and OCT2. Our results have reaffirmed previous conclusions regarding BOB1 selectivity towards the dimer configuration of OCT1/2. However, they suggest that the monomeric configuration of these factors, assembled on the classical octamer ATGCAAAT and related motifs, are the primary targets of BOB1. Our data further specify the DNA sequence preference imposed by BOB1 and predict the probability of ternary complex formation. These results provide an additional insight into the action of BOB1-an essential immune regulator and a promising molecular target for the treatment of autoimmune diseases and hematologic malignancies.