RESUMO
PIN1 is the only known enzyme capable of recognizing and isomerizing the phosphorylated Serine/ThreonineProline motif. Through this mechanism, PIN1 controls diverse cellular functions, including telomere maintenance. Both PIN1 overexpression and its involvement in oncogenic pathways are involved in several cancer types, including glioblastoma (GBM), a lethal disease with poor therapeutic resources. However, knowledge of the role of PIN1 in GBM is limited. Thus, the present work aimed to study the role of PIN1 as a telomere/telomerase regulator and its contribution to tumor biology. PIN1 knockout (KO) LN229 cell variant using CRISPR/Cas9 was developed and compared with PIN1 LN229 expressing cells. To study the effect of PIN1 absence, status of NFκB pathway was evaluated by luciferase reporter gene assay and quantitative PCR. Results revealed that PIN1 deletion in GBM cells diminished the active levels of NFκB and decrease the transcription of il8 and htert genes. Then, telomere/telomerase related processes were studied by RQTRAP assay and telomere length determination by qPCR, obtaining a reduction both in telomerase activity as in telomere length in PIN1 KO cells. In addition, measurement of SA ßgalactosidase and caspase3 activities revealed that loss of PIN1 triggers senescence and apoptosis. Finally, migration, cell cycle progression and tumorigenicity were studied by flow cytometry/western blot, Transwell assay and in vivo experiments, respectively. PIN1 deletion decreased migration as well as cell cycle progression by increasing doubling time and also resulted in the loss of LN229 cell ability to form tumors in mice. These results highlight the role of PIN1 in telomere homeostasis and GBM progression, which supports PIN1 as a potential molecular target for the development of novel therapeutic agents for GBM treatment.
Assuntos
Glioblastoma , Telomerase , Humanos , Animais , Camundongos , Glioblastoma/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Telomerase/metabolismo , Reação em Cadeia da Polimerase , Telômero/genética , Telômero/metabolismo , Linhagem Celular Tumoral , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismoRESUMO
To understand the mechanism of replication used by baculoviruses, it is essential to describe all the factors involved, including virus and host proteins and the sequences where DNA synthesis starts. A lot of work on this topic has been done, but there is still confusion in defining what sequence/s act in such functions, and the mechanism of replication is not very well understood. In this work, we performed an AgMNPV replication kinetics into the susceptible UFL-Ag-286 cells to estimate viral genome synthesis rates. We found that the viral DNA exponentially increases in two different phases that are temporally separated by an interval of 5 h, probably suggesting the occurrence of two different mechanisms of replication. Then, we prepared a plasmid library containing virus fragments (0.5-2 kbp), which were transfected and infected with AgMNPV in UFL-Ag-286 cells. We identified 12 virus fragments which acted as origins of replication (ORI). Those fragments are in close proximity to core genes. This association to the core genome would ensure vertical transmission of ORIs. We also predict the presence of common structures on those fragments that probably recruit the replication machinery, a structure also present in previously reported ORIs in baculoviruses.
Assuntos
Replicação do DNA , DNA Viral/genética , Genoma Viral , Nucleopoliedrovírus/genética , Nucleopoliedrovírus/fisiologia , Animais , Linhagem Celular , Cinética , Mariposas/virologia , Origem de Replicação , Replicação Viral/genéticaRESUMO
Telomeropathies involve a wide variety of infrequent genetic diseases caused by mutations in the telomerase maintenance mechanism or the DNA damage response (DDR) system. They are considered a family of rare diseases that often share causes, molecular mechanisms and symptoms. Generally, these diseases are not diagnosed until the symptoms are advanced, diminishing the survival time of patients. Although several related syndromes may still be unrecognized this work describes those that are known, highlighting that because they are rare diseases, physicians should be trained in their early diagnosis. The etiology and diagnosis are discussed for each telomeropathy and the treatments when available, along with a new classification of this group of diseases. Ethical and legal issues related to this group of diseases are also considered.
Assuntos
Dano ao DNA , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/terapia , Telomerase/genética , Homeostase do Telômero , Antecipação Genética , Ética Médica , Estudos de Associação Genética , Marcadores Genéticos , Testes Genéticos , Variação Genética , Humanos , Mutação , Fenótipo , Doenças Raras , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo , Homeostase do Telômero/genéticaRESUMO
Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.
Assuntos
Neoplasias/genética , Telomerase/genética , Telômero/fisiologia , Animais , Divisão Celular/fisiologia , Senescência Celular/genética , Humanos , Neoplasias/enzimologia , Telomerase/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologiaRESUMO
La telomerasa es la enzima responsable del mantenimiento de la longitud de los telómeros mediante la adición de secuencias repetitivas ricas en guanina, y su actividad se observa principalmente en gametos, células madre y células tumorales. En las células somáticas humanas el potencial de proliferación es limitado, alcanzando la senescencia luego de 50-70 divisiones celulares, debido a que la ADN polimerasa no es capaz de copiar el ADN en los extremos de los cromosomas. Por el contrario, en la mayoría de las células tumorales el potencial de replicación es ilimitado debido al mantenimiento de la longitud telomérica dado por la telomerasa. Los telómeros tienen proteínas adicionales que regulan la unión de la telomerasa. De la misma manera la telomerasa también se asocia con un complejo de proteínas que regulan su actividad. Este trabajo se centra en la estructura y función del complejo telómero/telomerasa y a cómo las alteraciones en su comportamiento conducen al desarrollo de diversas enfermedades, principalmente cáncer. El desarrollo de inhibidores del sistema telómero / telomerasa podría ser un blanco con posibilidades prometedoras.(AU)
Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.(AU)
Assuntos
Animais , Humanos , Neoplasias/genética , Telomerase/genética , Telômero/fisiologia , Senescência Celular/genética , Divisão Celular/fisiologia , Neoplasias/enzimologia , Telomerase/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologiaRESUMO
La telomerasa es la enzima responsable del mantenimiento de la longitud de los telómeros mediante la adición de secuencias repetitivas ricas en guanina, y su actividad se observa principalmente en gametos, células madre y células tumorales. En las células somáticas humanas el potencial de proliferación es limitado, alcanzando la senescencia luego de 50-70 divisiones celulares, debido a que la ADN polimerasa no es capaz de copiar el ADN en los extremos de los cromosomas. Por el contrario, en la mayoría de las células tumorales el potencial de replicación es ilimitado debido al mantenimiento de la longitud telomérica dado por la telomerasa. Los telómeros tienen proteínas adicionales que regulan la unión de la telomerasa. De la misma manera la telomerasa también se asocia con un complejo de proteínas que regulan su actividad. Este trabajo se centra en la estructura y función del complejo telómero/telomerasa y a cómo las alteraciones en su comportamiento conducen al desarrollo de diversas enfermedades, principalmente cáncer. El desarrollo de inhibidores del sistema telómero / telomerasa podría ser un blanco con posibilidades prometedoras.
Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.
Assuntos
Animais , Humanos , Neoplasias/genética , Telomerase/genética , Telômero/fisiologia , Senescência Celular/genética , Divisão Celular/fisiologia , Neoplasias/enzimologia , Telomerase/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , /fisiologiaRESUMO
Telomerase is the enzyme responsible for the maintenance of telomere length by adding guanine-rich repetitive sequences. Its activity can be seen in gametes, stem cells and tumor cells. In human somatic cells the proliferative potential is limited, reaching senescence after 50-70 cell divisions, because the DNA polymerase is not able to copy the DNA at the ends of chromosomes. By contrast, in most tumor cells the replicative potential is unlimited due to the maintenance of the telomeric length given by telomerase. Telomeres have additional proteins that regulate the binding of telomerase, likewise telomerase associates, with a protein complex that regulates its activity. This work focuses on the structure and function of the telomere/telomerase complex and how changes in its behavior lead to the development of different diseases, mainly cancer. Development of inhibitors of the telomere/telomerase complex could be a target with promising possibilities.
Assuntos
Neoplasias/genética , Telomerase/genética , Telômero/fisiologia , Animais , Senescência Celular/genética , Divisão Celular/fisiologia , Humanos , Neoplasias/enzimologia , Telomerase/metabolismo , Proteína 1 de Ligação a Repetições Teloméricas/fisiologia , Proteína 2 de Ligação a Repetições Teloméricas/fisiologiaRESUMO
BACKGROUND: Anticarsia gemmatalis is a pest in South America's soybean crops, which could be controlled by the Multinucleopolyhedrovirus of A. gemmatalis (AgMNPV). Currently, its commercial production is based on infected larvae. However, the possibility of using modified baculoviruses in Integrated Pest Management programs has stimulated an interest to develop alternative multiplication processes. This study evaluated the AgMNPV production in UFL-Ag-286 cells previously deprived Fetal Bovine Serum. RESULTS: Culture media containing 1% FBS during the previous 48 hours achieved a synchronized condition where 90% of cells were found in G0/G1 stage, showing the presence of non-filamentous actin. All characteristics were estimated from cellular viability tests, cell actin detection trials and flow cytometer cell cycle analysis. AgMNPV production was tested by transcript studies and budded viruses (BVs) and occlusion bodies (OBs) yield quantitation. Results showed that the productivity in FBS deprived cells was 9.8 times more in BVs and 3.8 times more in OBs with respect to non-treated cells. CONCLUSIONS: UFL-Ag-286 cells previously deprived in FBS shown to be a better host for AgMNPV propagation, increasing the useful for both in vitro bioinsecticide production and applications such as recombinant protein expression or gene delivery.