RESUMEN

Cancer cells have the ability to undergo an unlimited number of cell divisions, which gives them immortality. Thus, the cancer cell can extend the length of its telomeres, allowing these cells to divide unlimitedly and avoid entering the state of senescence or cellular apoptosis. One of the main effects of photobiomodulation (PBM) is the increase in the production of adenosine triphosphate (ATP) and free radicals, mainly reactive oxygen species (ROS). Existent data indicates that high levels of ROS can cause shortening and dysfunctional telomeres. Therefore, a better understanding of the effects induced by PBM on cancer cell telomere maintenance is needed. This work aimed to evaluate the effects of low-power red laser (658 nm) and blue LED (470 nm) on the TRF1 and TRF2 mRNA levels and telomere length in human breast cancer cells. MCF-7 and MDA-MB-231 cells were irradiated with a low-power red laser (69 J cm-2, 0.77 W/cm-2) and blue LED (482 J cm-2, 5.35 W/cm-2), alone or in combination, and the relative mRNA levels of the genes and telomere length were assessed by quantitative reverse transcription polymerase chain reaction. The results suggested that exposure to certain red laser and blue LED fluences decreased the TRF1 and TRF2 mRNA levels in both human breast cancer cells. Telomere length was increased in MCF-7 cells after exposure to red laser and blue LED. However, telomere length in MDA-MB-231 was shortened after exposure to red laser and blue LED at fluences evaluated. Our research suggests that photobiomodulation induced by red laser and low-power blue LED could alter telomere maintenance and length.

Asunto(s)

Neoplasias de la Mama , Terapia por Luz de Baja Intensidad , Telómero , Proteína 1 de Unión a Repeticiones Teloméricas , Proteína 2 de Unión a Repeticiones Teloméricas , Humanos , Neoplasias de la Mama/radioterapia , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Femenino , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Telómero/efectos de la radiación , Terapia por Luz de Baja Intensidad/métodos , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Línea Celular Tumoral , ARN Mensajero/metabolismo , ARN Mensajero/genética , Células MCF-7 , Homeostasis del Telómero/efectos de la radiación , Complejo Shelterina , Proteínas de Unión a Telómeros

RESUMEN

Muscle injuries are the most prevalent type of injury in sports. A great number of athletes have relapsed in muscle injuries not being treated properly. Photobiomodulation therapy is an inexpensive and safe technique with many benefits in muscle injury treatment. However, little has been explored about the infrared laser effects on DNA and telomeres in muscle injuries. Thus, the aim of this study was to evaluate photobiomodulation effects on mRNA relative levels from genes related to telomere and genomic stabilization in injured muscle. Wistar male rats were randomly divided into six groups: control, laser 25 mW, laser 75 mW, injury, injury laser 25 mW, and injury laser 75 mW. Photobiomodulation was performed with 904 nm, 3 J/cm2 at 25 or 75 mW. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly on the tibialis anterior muscle. After euthanasia, skeletal muscle samples were withdrawn and total RNA extracted for evaluation of mRNA levels from genomic (ATM and p53) and chromosome stabilization (TRF1 and TRF2) genes by real-time quantitative polymerization chain reaction. Data show that photobiomodulation reduces the mRNA levels from ATM and p53, as well reduces mRNA levels from TRF1 and TRF2 at 25 and 75 mW in injured skeletal muscle. In conclusion, photobiomodulation alters mRNA relative levels from genes related to genomic and telomere stabilization in injured skeletal muscle.

Asunto(s)

Cromosomas de los Mamíferos/genética , Genoma , Terapia por Luz de Baja Intensidad/métodos , Músculo Esquelético/lesiones , Músculo Esquelético/efectos de la radiación , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Proteínas Similares a la Proteína de Unión a TATA-Box/genética , Proteínas Similares a la Proteína de Unión a TATA-Box/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

BACKGROUND: Entamoeba histolytica is the protozoan parasite responsible for human amebiasis. It causes up to 100,000 deaths worldwide each year. This parasite has two closely related basal transcription factors, the TATA-box binding protein (EhTBP) and the TBP-related factor 1 (EhTRF1). TBP binds to the canonical TATTTAAA-box, as well as to different TATA variants. TRF1 also binds to the TATTTAAA-box. However, their binding capacity to diverse core promoter elements, including the GAAC-element, and their role in gene regulation in this parasite remains unknown. METHODS: EMSA experiments were performed to determine the binding capacity of recombinant TBP and TRF1 to TATA variants, GAAC and GAAC-like boxes. For the functional analysis under different stress stimuli (e.g. growth curve, serum depletion, heat-shock, and UV-irradiation) and during the interaction with mammalian cells (erythrocytes, MDCK cell monolayers, and hepatocytes of hamsters), RT-qPCR, and gene knockdown were performed. RESULTS: Both transcription factors bound to the different TATA variants tested, as well as to the GAAC-boxes, suggesting that they are GAAC-box-binding proteins. The K D values determined for TBP and TRF1 for the different TATA variants and GAAC-box were in the range of 10-12 M to 10-11 M. During the death phase of growth or in serum depletion, Ehtbp mRNA levels significantly increased, whereas the mRNA level of Ehtrf1 did not change under these conditions. Ehtrf1 gene expression was negatively regulated by UV-irradiation and heat-shock stress, with no changes in Ehtbp gene expression. Moreover, Ehtrf1 gene also showed a negative regulation during erythrophagocytosis, liver abscess formation, and a transient expression level increase at the initial phase of MDCK cell destruction. Finally, the Ehtbp gene knockdown displayed a drastic decrease in the efficiency of erythrophagocytosis in G3 trophozoites. CONCLUSIONS: To our knowledge, this study reveals that these basal transcription factors are able to bind multiple core promoter elements. However, their immediate change in gene expression level in response to different stimuli, as well as during the interaction with mammalian cells, and the diminishing of erythrophagocytosis by silencing the Ehtbp gene indicate the different physiological roles of these transcription factors in E. histolytica.

Asunto(s)

Proteínas Portadoras/genética , Regulación de la Expresión Génica , Proteína de Unión a TATA-Box/genética , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Factores de Transcripción/genética , Animales , Proteínas Portadoras/metabolismo , Clonación Molecular , Cricetinae , Perros , Entamoeba histolytica/genética , Técnicas de Silenciamiento del Gen , Hepatocitos/parasitología , Interacciones Huésped-Parásitos/genética , Células de Riñón Canino Madin Darby , Regiones Promotoras Genéticas , Reacción en Cadena en Tiempo Real de la Polimerasa , Estrés Fisiológico/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Transcripción Genética

RESUMEN

The objective of this study was to find the key regulatory molecules in the cell senescence process through observing the expression of telomere-associated factor during the normal cell replicative senescence process. Based on the established cell replicative senescence model, reverse transcription-polymerase chain reaction and western blot analyses were used to detect telomere-associated factor expression at the mRNA and protein levels, including that of human telomere binding protein 1, tankyrase 1, telomerase RNA, telomere protection protein 1 (POT1), and p53 during the process of human embryonic lung fibroblast replicative senescence. The results showed that transcription of human telomere binding protein 1 did not change with cell senescence, whereas the protein expression of human telomere binding protein 1 increased gradually and then decreased rapidly; there was no change in the mRNA and protein expression of POT1; with the replicative senescence of human embryonic lung fibroblasts, expression of POT1 decreased gradually; TRF1 showed an increasing trend with cell senescence; and p53 protein expression did not change. Together, the results from this study suggest that human telomere binding protein 1, POT1, and TRF1 played important roles in cell senescence.

Asunto(s)

Senescencia Celular/genética , Fibroblastos/metabolismo , Expresión Génica , Proteínas de Unión a Telómeros/genética , Línea Celular , Humanos , ARN/genética , Complejo Shelterina , Tanquirasas/genética , Tanquirasas/metabolismo , Telomerasa/genética , Proteínas de Unión a Telómeros/metabolismo , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 1 de Unión a Repeticiones Teloméricas/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo

RESUMEN

BACKGROUND: Telomeres are specialized structures at the end of chromosomes essential for maintaining genome stability and cell viability. The importance of telomeric proteins for telomere maintenance has increased our interest in the identification of homologues within the genus Leishmania. The mammalian TRF1 and TRF2 proteins, for example, bind double-stranded telomeres via a Myb-like DNA-binding domain and are involved with telomere length regulation and chromosome end protection. In addition, TRF2 can modulate the activity of several enzymes and influence the conformation of telomeric DNA. In this work, we identified and characterized a Leishmania protein (LaTRF) homologous to both mammalian TRF1 and TRF2. RESULTS: LaTRF was cloned using a PCR-based strategy. ClustalW and bl2seq sequence analysis showed that LaTRF shared sequence identity with the Trypanosoma brucei TRF (TbTRF) protein and had the same degree of sequence similarities with the dimerization (TRFH) and the canonical DNA-binding Myb-like domains of both mammalian TRFs. LaTRF was predicted to be an 82.5 kDa protein, indicating that it is double the size of the trypanosome TRF homologues. Western blot and indirect immunofluorescence combined with fluorescence in situ hybridization showed that LaTRF, similarly to hTRF2, is a nuclear protein that also associates with parasite telomeres. Native and full length LaTRF and a mutant bearing the putative Myb-like domain expressed in bacteria bound double-stranded telomeric DNA in vitro. Chromatin immunoprecipitation showed that LaTRF interacted specifically with telomeres in vivo. CONCLUSION: The nuclear localization of LaTRF, its association and co-localization with parasite telomeres and its high identity with TbTRF protein, support the hypothesis that LaTRF is a Leishmania telomeric protein.

Asunto(s)

Leishmania mexicana/química , Leishmania mexicana/fisiología , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , Proteínas de Unión a Telómeros/análisis , Proteínas de Unión a Telómeros/genética , Telómero/química , Secuencia de Aminoácidos , Western Blotting , Inmunoprecipitación de Cromatina , Clonación Molecular , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética

RESUMEN

Several studies revealed a similar down-regulation of telomeric repeat binding factor 1 (TRF1) in tumors. We have previously reported the TRFl expression levels were down-regulation in non-small cell lung cancer (NSCLC). The regulation of TRFl localization is proposed to be important for the function and expression. The nuclear localization signal (NLS) and nuclear export signal (NES) are often important clues to localization of protein. The objective of the present study was to investigate the NLS and NES of TRFl in NSCLC patients. Thirty (30) patients with NSCLCs had undergone radical operations in The First Affiliated Hospital, College of Medicine, Zhejiang University. DNA sequences of NLSs and NESs were amplified by PCR. The PCR products were analyzed by DNA sequencing. There were four NLSs of the TRFl protein, including two monopartite and two bipartite NLSs. The NLSs sequences were included in 337KKERRVGTPQSTKKKKESRR356. The exon 8 and exon 9 of TRFl DNA were covered the NLS sequences. The sequences of predicted NESs were 11WMLDFLCLSL86 and 174NLLKLQALAV183, respectively. The exon 1, exon 3 and exon 4 of TRFl were covered the NES sequences. In NSCLCs, there was no a mutation, deletion, or substitution in NLS and NES of TRFl. We conclude that the NLS and NES sequences in NSCLCs patients did not have mutations. Down-expression of TRFl does not indicate gene mutation of NLS and NES in NSCLCs.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación hacia Abajo/genética , Neoplasias Pulmonares/genética , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Exones , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , Señales de Exportación Nuclear/genética , Señales de Localización Nuclear/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Complejo Shelterina

RESUMEN

Several studies revealed a similar down-regulation of telomeric repeat binding factor 1 (TRF1) in tumors. We have previously reported the TRFl expression levels were down-regulation in non-small cell lung cancer (NSCLC). The regulation of TRFl localization is proposed to be important for the function and expression. The nuclear localization signal (NLS) and nuclear export signal (NES) are often important clues to localization of protein. The objective of the present study was to investigate the NLS and NES of TRFl in NSCLC patients. Thirty (30) patients with NSCLCs had undergone radical operations in The First Affiliated Hospital, College of Medicine, Zhejiang University. DNA sequences of NLSs and NESs were amplified by PCR. The PCR products were analyzed by DNA sequencing. There were four NLSs of the TRFl protein, including two monopartite and two bipartite NLSs. The NLSs sequences were included in 337KKERRVGTPQSTKKKKESRR356. The exon 8 and exon 9 of TRFl DNA were covered the NLS sequences. The sequences of predicted NESs were 11WMLDFLCLSL86 and 174NLLKLQALAV183, respectively. The exon 1, exon 3 and exon 4 of TRFl were covered the NES sequences. In NSCLCs, there was no a mutation, deletion, or substitution in NLS and NES of TRFl. We conclude that the NLS and NES sequences in NSCLCs patients did not have mutations. Down-expression of TRFl does not indicate gene mutation of NLS and NES in NSCLCs.

Asunto(s)

Femenino , Humanos , Masculino , Carcinoma de Pulmón de Células no Pequeñas/genética , Regulación hacia Abajo/genética , Neoplasias Pulmonares/genética , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión a Repeticiones Teloméricas/genética , Exones , Regulación Neoplásica de la Expresión Génica , Señales de Exportación Nuclear/genética , Señales de Localización Nuclear/genética , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN