RESUMO
Previously we've described the obtainment of a subpopulation of cancer stem cells from a human colorec- tal carcinoma cell line MIP101. These cells possess elevated clonogenic and tumorigenic capacities. According to our data, depletion of stem compartment in a cancer cell population blocks its tumorigenicity. The current work is dedicated to the comparison of tumorigenic potential between cell populations with enriched or depleted stem compartment. We show that tumor growth following xenografting of enriched stem cell population can be suppressed by intramuscular injections of ganciclovir. Thus, we report a method to obtain a cell population with high Oct4 promoter expression within the MIP101 colorectal carcinoma cell line and to eliminate these cells from the population in vitro as well as in vivo.
Assuntos
Biomarcadores Tumorais/biossíntese , Neoplasias Colorretais/patologia , Neoplasias Colorretais/prevenção & controle , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Fator 3 de Transcrição de Octâmero/biossíntese , Animais , Biomarcadores Tumorais/genética , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Ganciclovir/farmacologia , Ganciclovir/uso terapêutico , Vetores Genéticos , Humanos , Lentivirus/genética , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Puromicina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
An adult mammal is composed of more than 200 different types of specialized somatic cells whose differentiated state remains stable over the life of the organism. For a long time it was believed that the differentiation process is irreversible, and the transition between the two types of specialized cells is impossible. The possibility of direct conversion of one differentiated cell type to another was first shown in the 80s of the last century in experiments on the conversion of fibroblasts into myoblasts by ectopic expression of the transcription factor MyoD. Surprisingly, this technology has remained unclaimed in cell biology for a long time. Interest in it revived after 200 thanks to the research of Novel Prize winner Shinya Yamanaka who has shown that a small set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is capable of restoring pluripotency in somatic cells which they lost in the process of differentiation. In 2010, using a similar strategy and the tissue-specific transcription factors Vierbuchen and coauthors showed the possibility of direct conversion of fibroblasts into neurons, i. e. the possibility of transdifferentiation of one type of somatic cells in the other. The works of these authoras were a breakthrough in the field of cell biology and gave a powerful impulse to the development of cell technologies for the needs of regenerative medicine. The present review discusses the main historical discoveries that preceded this work, evaluates the status of the problem and the progress in the development of methods for reprogramming at the moment, describes the main approaches to solving the problems of reprogramming of somatic cells into neuronal, and briefly discusses the prospect of application of reprogramming and transdifferentiation of cells for such important application areas as regenerative medicine, cell replacement therapy and drug screening.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos/tendências , Reprogramação Celular/genética , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Neurônios/citologia , Fatores de Transcrição/genética , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Transdiferenciação Celular , Fibroblastos/citologia , Fibroblastos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Neurônios/metabolismo , Medicina Regenerativa , Fatores de Transcrição/metabolismoRESUMO
In the current work we make an attempt to compare cancer cells of one origin, but differing in the expression of CEA protein, a clinical marker of metastatic carcinomas, presumably one of the key factors in metastatic activity. We have explored the morphology of cell colonies in vitro, expression patterns of epithelial markers, the ability of these cells to form tumors and metastases in vivo, and evaluated their stem compartment with the aid of a suicidal genetic construct sensitive to the embryonic stem cell marker, Oct4.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/secundário , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/genética , Animais , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Células Clonais , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Carga TumoralRESUMO
Investigations of transcriptional regulation of Oct4 gene in mouse embryonic stem cells have revealed an important cis-element--the distal enhancer (DE). DE consists of two functionally significant elements--DEa and DEb. Both elements are necessary to complete the DE-mediated expression of Oct4 gene in pluripotent cells. The most likely candidates for the binding site DEb are Oct4 itself in complex with Sox2 protein. It remains unclear which transcriptional proteins bind to the DEa site and what is the mechanism of the co-operation between the DEa and the DEb. Through the use of using the EMSA and chromatographic fractionation of proteins from extracts of mouse embryonic stem cells and mouse tissues, were isolated proteins specifically interacting with the sequence DEa Oct4 gene.
Assuntos
Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Química Encefálica , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Camundongos , Dados de Sequência Molecular , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/genética , Ligação Proteica , Fatores de Transcrição SOXB1/genética , Transdução de SinaisRESUMO
In present publication we describe for the first time the obtainment of cancer stem cells from a weakly metastatic human colorectal carcinoma cell line MIP101 via selecting from the native population the cells that express intensively an embryonic stem cell marker, POU5F1 (Oct4). We provide the evidence that these cells possess an elevated clonogenic and tumorigenic potential when compared to the native population, and this correlates to the hypothesis of cancer stem cells' primary role in the development of malignant neoplasms.
Assuntos
Neoplasias Colorretais/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Neoplásicas/citologia , Fator 3 de Transcrição de Octâmero/biossíntese , Animais , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Células-Tronco Embrionárias/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Metástase Neoplásica/genética , Metástase Neoplásica/patologia , Células-Tronco Neoplásicas/metabolismoRESUMO
Previously, our results of a two-hybrid screening essay allowed us to recognize p68 (DDX5) as a possible partner of CDX2. The recent part of research was carried out to confirm this interaction. We show the co-localization of these proteins in the nuclei of colon carcinoma cell line and of epithelium of villi. By means of GST-pulldown we reveal DDX5 as a part of a complex with CDX2. During the investigation of the effect of DDX5-CDX2 interaction upon beta-catenin-mediated transcription regulation we note that in each of three investigated cell lines Cdx2 acts as an activator of luciferase expression. In T98G and U20S cell lines we observe a partial decline of beta-catenin transcription enhancing effect while interacting with CDX2. In the cell systems studied, DDX5 acts as a weak repressor both solely and together with CDX2 and beta-catenin. Concerning the influence upon D1 cyclin promoter, we find that, depending on environment, CDX2 may either decline its transcription (U20S line) or raise it (T98G). Besides, PDGF reduces CDX2 activity both in activation and repression. When DDX5 and CDX2 are transfected in T98G cells together, the repressing activity of DDX5 is leveled with activation by Cdx2. In both cell lines the native DDX5 acts as a weak repressor of D1 cycline; PDGF treatment does no significant effect on its activity.
Assuntos
Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , Proteínas de Homeodomínio/metabolismo , Regiões Promotoras Genéticas/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica/fisiologia , Fator de Transcrição CDX2 , Células CACO-2 , Núcleo Celular/genética , Ciclina D1/biossíntese , Ciclina D1/genética , RNA Helicases DEAD-box/genética , Células HEK293 , Proteínas de Homeodomínio/genética , Humanos , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fatores de Transcrição/genética , beta Catenina/biossíntese , beta Catenina/genéticaRESUMO
The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.
Assuntos
Desdiferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/citologia , Animais , Linhagem Celular , Meios de Cultura , Células-Tronco Pluripotentes Induzidas/metabolismo , Lentivirus , Camundongos , Ratos , Transdução Genética/métodosRESUMO
Induced pluripotent stem (iPS) cells are derived from somatic cells reprogrammed to the pluripotent state by the induced expression of defined transcription factors, achieved for the first time by the seminal work of Takahashi and Yamanaka. This new type of pluripotent cells has offered new exciting options in regenerative medicine allowing the replacement of cells and organs with the patient's own cells thereby avoiding immunological complications. In order to develop such technologies in approved animal models, iPS cells were also generated from rodents. Of course, the most important model for studying of different diseases is rat. In this study, we present a method suitable for rat iPS cells genetic modification by stable transfection and show necessary conditions for the first stages of direct differentiation.
Assuntos
Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Linhagem Celular , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Ratos , Medicina Regenerativa/métodos , Transfecção/métodosRESUMO
A perspective of using embryonic stem (ES) and induced pluripotent stem (iPS) cells in clinical medicine makes karyological analysis of these cells an important issue. Using methods of classical and molecular cytogenetics chromosomal analysis, we have carried out karyological study of two mouse ES and two iPS cell lines derived de novo. We have found monosomy of X chromosome in all studied ES and iPS cell lines, thus making a modal number of chromosomes in these cell lines 39. A chromosomal instability (aneuploidy) was revealed in both studied iPS cell lines. Moreover, we have detected chromosomal rearrangements and chromosomal fragments in one of iPS cell line. Our findings underline the importance of careful cytogenetic evaluation of pluripotent cell lines, especially iPS cell lines, which should be carried out prior to any clinical use of these cells.