RESUMEN
Patients with neurological diseases, such as schizophrenia, tend to show low K+-Cl- co-transporter 2 (KCC2) levels in the brain. The cause of these diseases has been associated with stress and neuroinflammation. However, since the pathogenesis of these diseases is not yet fully investigated, drug therapy is still limited to symptomatic therapy. Targeting KCC2, which is mainly expressed in the brain, seems to be an appropriate approach in the treatment of these diseases. In this review, we aimed to discuss about stress and inflammation, KCC2 and Gamma-aminobutyric acid (GABA) function, diseases which decrease the KCC2 levels in the brain, factors that regulate KCC2 activity, and the possibility to overcome neuronal dysfunction targeting KCC2. We also aimed to discuss the relationships between neurological diseases and LPS caused by Porphyromonas gingivalis (P. g), which is a type of oral bacterium. Clinical trials on oxytocin, sirtuin 1 (SIRT1) activator, and transient receptor potential cation channel subfamily V Member 1 activator have been conducted to develop effective treatment methods. We believe that KCC2 modulators that regulate mitochondria, such as oxytocin, glycogen synthase kinase 3ß (GSK3ß), and SIRT1, can be potential targets for neurological diseases.
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To understand the molecular mechanisms responsible for radioresistance in cancer cells, we previously established clinically relevant radioresistant (CRR) cell lines from several human cancer cell lines. These CRR cells proliferate even under exposure to 2 Gy/day of X-rays for more than 30 days, which is a standard protocol for tumor radiotherapy. CRR cells received 2 Gy/day of X-rays to maintain their radioresistance (maintenance irradiation; MI). Interestingly, CRR cells that did not receive MI for more than a year lost their radioresistance, indicating that radiation-induced radioresistance is reversible. We designated these CRR-NoIR cells. Karyotyping of the parental and CRR cells revealed that the chromosomal composition of CRR cells is quite different from that of the parental cells. However, CRR and CRR-NoIR cells were more similar compared with the parental cells because CRR cells repair X-ray-induced DNA damage with higher fidelity. To identify the factor(s) involved in tumor radioresistance, previously published studies including ours have compared radioresistant cells to parental cells. In this review, we conclude that a comparison between CRR and CRR-NoIR cells, rather than parental cells, is the best way to identify factors involved in tumor radioresistance.
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Neoplasias , Tolerancia a Radiación , Humanos , Línea Celular Tumoral , Tolerancia a Radiación/genética , Rayos X , Daño del ADN , Neoplasias/genética , Neoplasias/radioterapiaRESUMEN
Auger electrons can induce nanoscale physiochemical damage to DNA. The present study reports a sequential and systematic evaluation of the relationship between DNA damage such as double-strand breaks (DSBs) and the cell cycle for the Auger electron-emitting agent radiolabeled cisplatin with DNA binding ability. For dynamic imaging analysis, we used U2OS-derived cancer cells expressing two fluorescent fusion proteins: tumor-suppressor p53 binding protein 1 with a green fluorescent protein (53BP1-EGFP) and proliferating cell nuclear antigen with a red fluorescent protein (PCNA-DsRed). Time-lapse images of the cells were quantitatively analyzed using the ImageJ software with the deepImageJ plugin and the Google Colaboratory platform. From the middle-to-late G1 phase, around the G1-to-S phase transition, we found increased 53BP1 foci in cells treated with the radio-cisplatin. The radio-cisplatin caused significantly more DSBs than the nonradioactive cisplatin and saline in the G1 phase but not in the other phases. These results indicate that Auger electron-induced DNA damage, including DSBs, depends on the cell cycle. The G1 phase, which is associated with low DNA repair capacity and high radiosensitivity, is a promising target; thus, combining radiolabeled cisplatin with agents that arrest cells in the G1 phase could improve the DNA-damaging effect of Auger electrons and their therapeutic efficacy.
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Cisplatino , Electrones , Cisplatino/farmacología , División Celular , Ciclo Celular , Daño del ADNRESUMEN
Cranial radiation therapy (CRT) is an effective treatment for brain tumors; however, it also causes brain injuries. The pediatric brain is considered especially vulnerable compared to the adult brain; thus, brain injuries caused by CRT may severely affect their quality of life. In this study, we determined the neuroprotective effects of nasal oxytocin administration following cranial radiation in mice. We investigated the cognitive behavior of mice (novel object recognition test and novel object location test), phosphorylated histone H2AX (γ-H2AX) and K+-Cl- transporter (KCC2) by immunohistochemical analysis of the hippocampal sections, and neuronal cells by immunocytochemistry after radiation and oxytocin administration. We found that the number of γ-H2AX foci was increased, and the surface signal intensity of KCC2 immunofluorescence was decreased in cells that were irradiated with X-rays (1.5 Gy, for three consecutive days) compared with cells that were not. Furthermore, using MQAE, we found that the intracellular chloride ion concentration was downregulated in oxytocin-treated cells by increasing surface KCC2 expression. These results indicate that nasal oxytocin administration after cranial irradiation attenuates cognitive dysfunction in mice and exerts multifaceted neuroprotective effects on DNA damage and maintains chloride ion concentration in neuronal cells.
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Lesiones Encefálicas , Disfunción Cognitiva , Fármacos Neuroprotectores , Simportadores , Animales , Lesiones Encefálicas/metabolismo , Cloruros/metabolismo , Disfunción Cognitiva/metabolismo , Irradiación Craneana/métodos , Daño del ADN , Hipocampo/metabolismo , Humanos , Ratones , Fármacos Neuroprotectores/metabolismo , Fármacos Neuroprotectores/farmacología , Oxitocina/metabolismo , Oxitocina/farmacología , Calidad de Vida , Simportadores/metabolismoRESUMEN
This study aims to establish new labeling methods for no-carrier-added radio-Pt (191Pt) and to evaluate the in vitro properties of 191Pt-labeled agents compared with those of agents labeled with the common emitter 111In. 191Pt was complexed with the DNA-targeting dye Hoechst33258 via diethylenetriaminepentaacetic acid (DTPA) or the sulfur-containing amino acid cysteine (Cys). The intranuclear fractions of 191Pt- and 111In-labeled Hoechst33258 were comparable, indicating that the labeling for 191Pt via DTPA or Cys and the labeling for 111In via DTPA worked equally well. 191Pt showed a DNA-binding/cellular uptake ratio of more than 1 order of magnitude greater than that of 111In. [191Pt]Pt-Hoechst33258 labeled via Cys showed a higher cellular uptake than that labeled via DTPA, resulting in a very high DNA-binding fraction of [191Pt]Pt-Cys-Hoechst33258 and extensive DNA damage. Our labeling methods of radio-Pt, especially via Cys, promote the development of radio-Pt-based agents for use in Auger electron therapy targeting DNA.
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Cisteína , Ácido Pentético , Cisteína/química , ADN , Electrones , Ácido Pentético/químicaRESUMEN
Inflammation, especially neuroinflammation, which is caused by stress, leads to central nervous system (CNS) dysfunction. Because lipopolysaccharides (LPSs) cause neuroinflammation, we investigated the effect of LPSs to CNS. In PC-12 cells, LPSs derived from oral bacteria reduced the expression of KCC2, a Cl- transporter. LPS derived from P. gingivalis (P. g) administered to rat primary cultured cells also reduced the KCC2 expression. However, LPSs derived from E. coli did not reduce the KCC2 expression. LPS treatment activated TLR4, IL-1ß, and REST gene expressions, which led to KCC2 inactivation in PC-12 cells. The mechanism of KCC2 has been shown to play an important role in brain maturation, function (such as the GABA switch), and behavioral problems, we investigated the GABA function. We found that the GABA function was changed from inhibitory to excitatory by the LPS derived from P. g treatment. We demonstrated that the GSK3ß also involved in the KCC2 reduction by LPS treatment. We show that oxytocin rescued the reduction in KCC2 expression caused by LPSs by inhibiting GSK3ß signaling but vasopressin could not. Considered together, our results indicate that the LPSs from oral bacteria but not the LPS from E. coli increase the risk for brain disorders and oxytocin might be a candidate to overcome the abnormal behavior caused by brain disorders such as psychiatric disorders.
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Encefalopatías , Simportadores , Animales , Células Cultivadas , Escherichia coli/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Lipopolisacáridos/toxicidad , Oxitocina/metabolismo , Oxitocina/farmacología , Células PC12 , Ratas , Simportadores/genética , Simportadores/metabolismo , Ácido gamma-AminobutíricoRESUMEN
AIMS: To overcome radioresistant cancer cells, clinically relevant radioresistant (CRR) cells were established. To maintain their radioresistance, CRR cells were exposed 2 Gy/day of X-rays daily (maintenance irradiation: MI). To understand whether the radioresistance induced by X-rays was reversible or irreversible, the difference between CRR cells and those without MI for a year (CRR-NoIR cells) was investigated by the mitochondrial function as an index. MAIN METHODS: Radiation sensitivity was determined by modified high density survival assay. Mitochondrial membrane potential (Δψm) was determined by 5,5',6,6'-tetrachloro-1,1', tetraethylbenzimidazolocarbo-cyanine iodide (JC-1) staining. Rapid Glucose-Galactose assay was performed to determine the shift in their energy metabolism from aerobic glycolysis to oxidative phosphorylation in CRR cells. Involvement of prohibitin-1 (PHB1) in Δψm was evaluated by knockdown of PHB1 gene followed by real-time PCR. KEY FINDINGS: CRR cells that exhibited resistant to 2 Gy/day X-ray lost their radioresistance after more than one year of culture without MI for a year. In addition, CRR cells lost their radioresistance when the mitochondria were activated by galactose. Furthermore, Δψm were increased and PHB1 expression was down-regulated, in the process of losing their radioresistance. SIGNIFICANCE: Our finding reveled that tune regulation of mitochondrial function is implicated in radioresistance phenotype of cancer cells. Moreover, as our findings indicate, though further studies are required to clarify the precise mechanisms underlying cancer cell radioresistance, radioresistant cells induced by irradiation and cancer stem cells that are originally radioresistant should be considered separately, the radioresistance of CRR cells is reversible.
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Potencial de la Membrana Mitocondrial/fisiología , Membranas Mitocondriales/metabolismo , Tolerancia a Radiación/fisiología , Biomarcadores Farmacológicos , Línea Celular Tumoral , Supervivencia Celular/genética , Humanos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Membranas Mitocondriales/fisiología , Neoplasias/metabolismo , Células Madre Neoplásicas , Tolerancia a Radiación/efectos de la radiación , Rayos X/efectos adversosRESUMEN
Mitochondria are very important intracellular organelles because they have various functions. They produce ATP, are involved in cell signaling and cell death, and are a major source of reactive oxygen species (ROS). Mitochondria have their own DNA (mtDNA) and mutation of mtDNA or change the mtDNA copy numbers leads to disease, cancer chemo/radioresistance and aging including longevity. In this review, we discuss the mtDNA mutation, mitochondrial disease, longevity, and importance of mitochondrial dysfunction in cancer first. In the later part, we particularly focus on the role in cancer resistance and the mitochondrial condition such as mtDNA copy number, mitochondrial membrane potential, ROS levels, and ATP production. We suggest a therapeutic strategy employing mitochondrial transplantation (mtTP) for treatment-resistant cancer.
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ADN Mitocondrial/fisiología , Longevidad/fisiología , Mitocondrias/fisiología , Mutación , Neoplasias/terapia , Adenosina Trifosfato/metabolismo , Trasplante de Células/métodos , ADN Mitocondrial/genética , Humanos , Mitocondrias/trasplante , Enfermedades Mitocondriales/genética , Neoplasias/metabolismo , Neoplasias/patología , Tolerancia a Radiación/genéticaRESUMEN
In cancer therapy, radioresistance or chemoresistance cells are major problems. We established clinically relevant radioresistant (CRR) cells that can survive over 30 days after 2 Gy/day X-ray exposures. These cells also show resistance to anticancer agents and hydrogen peroxide (H2O2). We have previously demonstrated that all the CRR cells examined had up-regulated miR-7-5p and after miR-7-5p knockdown, they lost radioresistance. However, the mechanism of losing radioresistance remains to be elucidated. Therefore, we investigated the role of miR-7-5p in radioresistance by knockdown of miR-7-5p using CRR cells. As a result, knockdown of miR-7-5p increased reactive oxygen species (ROS), mitochondrial membrane potential, and intracellular Fe2+ amount. Furthermore, miR-7-5p knockdown results in the down-regulation of the iron storage gene expression such as ferritin, up-regulation of the ferroptosis marker ALOX12 gene expression, and increases of Liperfluo amount. H2O2 treatment after ALOX12 overexpression led to the enhancement of intracellular H2O2 amount and lipid peroxidation. By contrast, miR-7-5p knockdown seemed not to be involved in COX-2 and glycolysis signaling but affected the morphology of CRR cells. These results indicate that miR-7-5p control radioresistance via ROS generation that leads to ferroptosis.
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Ferroptosis , Regulación Neoplásica de la Expresión Génica , MicroARNs/genética , Neoplasias de la Boca/patología , Tolerancia a Radiación , Especies Reactivas de Oxígeno/metabolismo , Araquidonato 12-Lipooxigenasa/metabolismo , Células HeLa , Humanos , Peróxido de Hidrógeno/metabolismo , Potencial de la Membrana Mitocondrial , Neoplasias de la Boca/genética , Neoplasias de la Boca/radioterapia , Transducción de Señal , Células Tumorales CultivadasRESUMEN
BACKGROUND: Radiation therapy is a highly cost-effective treatment for cancer, but the existence of radio-resistant cells remains the most critical obstacle in radiotherapy. We have been established clinically relevant radioresistant (CRR) cell lines by exposure to a stepwise increase of fractionated X-rays. We are trying to overcome the radio-resistance by analyzing the properties of these cells. In this study, we tried to evaluate the effects of hydrogen peroxide (H2O2) on the CRR cells because this can evaluate the efficacy of Kochi Oxydol-Radiation Therapy for Unresectable Carcinomas (KORTUC) that treats H2O2 before irradiation. We also established H2O2-resistant cells to compare the radiation and H2O2 resistant phenotype. MATERIALS AND METHODS: We used human cancer cell lines derived from hepatoblastoma (HepG2), oral squamous cell carcinoma (SAS), and cervical cancer (HeLa). We established HepG2, SAS, and HeLa CRR cells and HepG2, SAS, and HeLa H2O2-resistant cells. To evaluate their sensitivity to radiation or H2O2, high-density survival assay, or WST assay was performed. CellROXTM was used to detect intracellular Reactive Oxygen Species (ROS). RESULTS: CRR cells were resistant to H2O2-induced cell death but H2O2-resistant cells were not resistant to irradiation. This phenotype of CRR cells was irreversible. The intracellular ROS was increased in parental cells after H2O2 treatment for 3 h, but in CRR cells, no significant increase was observed. CONCLUSION: Fractionated X-ray exposure induces H2O2 resistance in CRR cells. Therefore, it is necessary to carry out cancer therapy such as KORTUC with the presence of these resistant cells in mind, and as the next stage, it would be necessary to investigate the appearance rate of these cells immediately and take countermeasures.
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Peróxido de Hidrógeno/farmacología , Tolerancia a Radiación/efectos de los fármacos , Radiación Ionizante , Muerte Celular/efectos de los fármacos , Muerte Celular/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Humanos , Especies Reactivas de Oxígeno/metabolismo , Rayos XRESUMEN
MicroRNA (miRNA) is a non-coding RNA involved in regulating both cancer gene promotion and suppression. We investigated the role of miRNA in inducing radiation resistance in cancer cell lines using clinically relevant radioresistant (CRR) cells. Analysis using miRNA arrays and qPCR revealed that miR-7-5p is highly expressed in all examined CRR cells. Additionally, CRR cells lose their radioresistance when daily irradiation is interrupted for over 6 months. MiR-7-5p expression is reduced in these cells, and treating CRR cells with a miR-7-5p inhibitor leads to a loss of resistance to irradiation. Conversely, overexpression of miR-7-5p in CRR cells using a miR-7-5p mimic shows further resistance to radiation. Overexpression of miR-7-5p in parent cells also results in resistance to radiation. These results indicate that miR-7-5p may control radioresistance in various cancer cells at the clinically relevant dose of irradiation. Furthermore, miR-7-5p downregulates mitoferrin and reduces Fe2+, which influences ferroptosis. Our findings have great potential not only for examining radiation resistance prior to treatment but also for providing new therapeutic agents for treatment-resistant cancers.
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Espacio Intracelular/metabolismo , Hierro/metabolismo , MicroARNs/genética , Tolerancia a Radiación/efectos de la radiación , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Células HeLa , Células Hep G2 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Interferencia de ARN , Tolerancia a Radiación/genéticaRESUMEN
4-Hydroxynonenal (HNE) is an important product of plasma membrane lipid peroxidation, which is a cause of cell and tissue injury. Mitochondrial DNA (mtDNA)-depleted ρ0 cells were established using human cervical cancer and oral squamous cell carcinoma cell lines. We investigated the effect of reactive oxygen species in ρ0 cells, especially the mechanism of hydrogen peroxide (H2 O2 )-mediated cell death. These cell were subjected to high oxidative stress and, compared with their parental cells, showed greater sensitivity to H2 O2 and high lipid peroxidation. Upregulation of HNE in the plasma membrane was observed prior to the increase in intracellular H2 O2 . The amount of oxidized lipid present changed H2 O2 permeability and administration of oxidized lipid led to further cell death after treatment with H2 O2 . Expression levels of lipoxygenase ALOX genes (ie ALOX5, ALOX12, and ALOX15) were upregulated in ρ0 cells, as were expression levels of ALOX12 and ALOX15 proteins. ALOX5 protein was mainly distributed in the nucleus, while ALOX12 and ALOX15 proteins were distributed in the nucleus and the cytoplasm. Although expression of COX2 gene was upregulated, its protein expression did not increase. ALOX (especially ALOX15) may be involved in the sensitivity of cancer cells to treatment. These data offer promise for the development of novel anticancer agents by altering the oxidation state of the plasma membrane. Our results showed that lipid peroxidation status is important for H2 O2 sensitivity and that ALOX15 is involved in lipid peroxidation status.
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Apoptosis/efectos de los fármacos , Permeabilidad de la Membrana Celular/genética , ADN Mitocondrial/genética , Peróxido de Hidrógeno/administración & dosificación , Peroxidación de Lípido/genética , Neoplasias/patología , Aldehídos/metabolismo , Araquidonato 15-Lipooxigenasa/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Resistencia a Antineoplásicos , Proteínas del Complejo de Cadena de Transporte de Electrón/genética , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacocinética , Mitocondrias/genética , Mitocondrias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Estrés Oxidativo/efectos de los fármacos , Éteres Fosfolípidos/administración & dosificación , Regulación hacia ArribaRESUMEN
We present data about mitochondrial DNA (mtDNA) copy number and aquaporin (AQP) gene expression in clinically radioresistant (CRR), ρ0, and their parental cells from human cervical cancer and human tongue squamous cell carcinoma. In both ρ0 and CRR cells, the mtDNA copy number was lower than for the parental strain. In addition, the obtained data suggest an association between the gene expression levels of AQP (1, 3, 8, and 9) and the difference in hydrogen peroxide (H2O2) sensitivity between ρ0 and CRR cells. Here, the composition of cell culture medium differs between CRR and ρ0 cells. To compare the gene expression of AQPs between ρ0 and CRR cells, therefore, we showed the data as the ratio to that in their parental cells.
RESUMEN
Radiotherapy (RT) is one of the major modalities for the treatment of human cancer and has been established as an excellent local treatment for malignant tumors. However, the existence of radioresistant cells remains one of the most critical obstacles in RT. To know the characteristics of radioresistant cells, clinically relevant radioresistant (CRR) cell lines were established. CRR cells can continue to proliferate in vitro and in vivo after exposure to 2 Gy/day of X-rays for more than 30 days. Daily microscopic observation of the irradiated CRR cells has indicated that the increase in cell death is not observed within 7 days of irradiation with 10 Gy of X-rays, suggesting that cell death is involved in cellular radioresistance. Radiation-induced regulated cell death (RCD) can be classified into three categories: apoptosis, autophagy-dependent cell death and necrosis (necroptosis). This review focuses on an aspect of radiation-induced RCD that has often been neglected: the manner in which the cells are destroyed. In many studies, apoptosis is considered the primary mode of RCD in irradiated cancer cells; however, it is necessary to consider necrosis or necroptosis as one of the modes of radiation-induced RCD.
Asunto(s)
Neoplasias/patología , Neoplasias/radioterapia , Tolerancia a Radiación/efectos de la radiación , Animales , Muerte Celular/efectos de la radiación , HumanosRESUMEN
Radiation therapy is one of the choices to treat malignant tumors. In radiation therapy, existence of radiation-resistant cell is a major problem to overcome. We established clinically relevant radioresistant cells that had been obtained by exposing to 2 Gy/day X-rays for more than 30 days. These cells are resistant to 2 Gy/day X-ray exposure and anticancer agents. However, the underlying resistance mechanism remains unclear. We investigated the resistance of clinically relevant radioresistant cells to hydrogen peroxide (H2O2), confirming a degree of resistance. Neither catalase enzyme activity nor aquaporins appeared to be involved in H2O2 resistance. Mitochondrial DNA copy number, adenosine triphosphate (ATP) concentration, and plasma membrane potential were decreased. The timing of H2O2 intake was delayed and lipid peroxidation was decreased. Sensitivity of clinically relevant radioresistant cells to H2O2 was enhanced by 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine administration. These results suggest that the membrane status is a major factor conferring H2O2 resistance in clinically relevant radioresistant cells, and we should further investigate how membrane status could be used to enhance the therapeutic effect on cancer.
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Antineoplásicos/farmacología , Carcinoma de Células Escamosas/patología , Peróxido de Hidrógeno/farmacología , Peroxidación de Lípido/efectos de los fármacos , Neoplasias de la Boca/patología , Oxidantes/farmacología , Tolerancia a Radiación/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/radioterapia , Catalasa/metabolismo , Células HeLa , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Neoplasias de la Boca/tratamiento farmacológico , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/radioterapia , Oxidación-Reducción , Células Tumorales Cultivadas , Rayos XRESUMEN
Radiotherapy (RT) is one of the major modalities for the treatment of human cancers and has been established as an excellent local treatment for malignant tumors. Conventional fractionated RT consists of 2-Gy X-rays, fractionated once a day, 5 days a week for 5-7 weeks in total 60 Gy. The efficacy of RT depends on the existence of radioresistant cells, which remains one of the most critical obstacles in RT and radio-chemotherapy. To improve the efficacy of RT, understanding the characteristics of radioresistant cells is one of the important subjects in radiation biology. Several studies have been reported to find out molecules implicated in radioresistance. However, it is noteworthy that cellular radioresistance has been mainly studied among cells with different genetic backgrounds and different origins. Therefore, making a system to compare between radioresistant and sensitive cells with the isogenic background is required. In this review, some aspects of cellular radioresistance mainly focusing on clinically relevant radioresistant (CRR) cell lines that can continue to proliferate even under exposure to 2-Gy X-rays, once a day, for more than 30 days, which is consistent with the conventional fractionated RT are discussed.
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Muerte Celular/efectos de la radiación , Modelos Biológicos , Tolerancia a Radiación/genética , Animales , Muerte Celular/genética , Línea Celular Tumoral , Supervivencia Celular/genética , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Humanos , Ratones , Ratones Desnudos , Dosis de Radiación , Rayos X , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To clarify the relationship between mitochondrial DNA (mtDNA)-depleted ρ0 cells and the cellular sensitivity to hydrogen peroxide (H2O2), we established HeLa and SAS ρ0 cell lines and investigated their survival rate in H2O2, radical scavenging enzymes, plasma membrane potential status, and chronological change in intracellular H2O2 amount under the existence of extracellular hydrogen peroxide compared with the parental cells. The results revealed that ρ0 cells had higher sensitivity to H2O2 than their parental cells, even though the catalase activity of ρ0 cells was up-regulated, and the membrane potential of the ρ0 cells was lower than their parental cells. Furthermore, the internal H2O2 amount significantly increased only in ρ0 cells after 50 µM H2O2 treatment for 1 h. These results suggest that plasma membrane status of ρ0 cells may cause degradation, and the change could lead to enhanced membrane permeability to H2O2. As a consequence, ρ0 cells have a higher H2O2 sensitivity than the parental cells.
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Membrana Celular/efectos de los fármacos , ADN Mitocondrial/efectos de los fármacos , Peróxido de Hidrógeno/farmacología , Membrana Celular/metabolismo , Proliferación Celular/efectos de los fármacos , ADN Mitocondrial/metabolismo , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The catalytic subunit of DNA dependent protein kinase (DNA-PKcs) and its kinase activity are critical for mediation of non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSB) in mammalian cells after gamma-ray irradiation. Additionally, DNA-PKcs phosphorylations at the T2609 cluster and the S2056 cluster also affect DSB repair and cellular sensitivity to gamma radiation. Previously we reported that phosphorylations within these two regions affect not only NHEJ but also homologous recombination repair (HRR) dependent DSB repair. In this study, we further examine phenotypic effects on cells bearing various combinations of mutations within either or both regions. Effects studied included cell killing as well as chromosomal aberration induction after 0.5-8 Gy gamma-ray irradiation delivered to synchronized cells during the G0/G1 phase of the cell cycle. Blocking phosphorylation within the T2609 cluster was most critical regarding sensitization and depended on the number of available phosphorylation sites. It was also especially interesting that only one substitution of alanine in each of the two clusters separately abolished the restoration of wild-type sensitivity by DNA-PKcs. Similar patterns were seen for induction of chromosomal aberrations, reflecting their connection to cell killing. To study possible change in coordination between HRR and NHEJ directed repair in these DNA-PKcs mutant cell lines, we compared the induction of sister chromatid exchanges (SCEs) by very low fluencies of alpha particles with mutant cells defective in the HRR pathway that is required for induction of SCEs. Levels of true SCEs induced by very low fluence of alpha-particle irradiation normally seen in wild-type cells were only slightly decreased in the S2056 cluster mutants, but were completely abolished in the T2609 cluster mutants and were indistinguishable from levels seen in HRR deficient cells. Again, a single substitution in the S2056 together with a single substitution in the T2609 cluster abolished SCE formation and thus also effectively interferes with HRR.
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Partículas alfa/efectos adversos , Proteína Quinasa Activada por ADN/metabolismo , Fase G1/efectos de la radiación , Rayos gamma/efectos adversos , Fase de Descanso del Ciclo Celular/efectos de la radiación , Serina/metabolismo , Treonina/metabolismo , Animales , Células CHO , Aberraciones Cromosómicas/efectos de la radiación , Cricetinae , Cricetulus , Proteína Quinasa Activada por ADN/químicaRESUMEN
We have examined cell-cycle dependence of chromosomal aberration induction and cell killing after high or low dose-rate γ irradiation in cells bearing DNA-PKcs mutations in the S2056 cluster, the T2609 cluster, or the kinase domain. We also compared sister chromatid exchanges (SCE) production by very low fluences of α-particles in DNA-PKcs mutant cells, and in homologous recombination repair (HRR) mutant cells including Rad51C, Rad51D, and Fancg/xrcc9. Generally, chromosomal aberrations and cell killing by γ-rays were similarly affected by mutations in DNA-PKcs, and these mutant cells were more sensitive in G1 than in S/G2 phase. In G1-irradiated DNA-PKcs mutant cells, both chromosome- and chromatid-type breaks and exchanges were in excess than wild-type cells. For cells irradiated in late S/G2 phase, mutant cells showed very high yields of chromatid breaks compared to wild-type cells. Few exchanges were seen in DNA-PKcs-null, Ku80-null, or DNA-PKcs kinase dead mutants, but exchanges in excess were detected in the S2506 or T2609 cluster mutants. SCE induction by very low doses of α-particles is resulted from bystander effects in cells not traversed by α-particles. SCE seen in wild-type cells was completely abolished in Rad51C- or Rad51D-deficient cells, but near normal in Fancg/xrcc9 cells. In marked contrast, very high levels of SCEs were observed in DNA-PKcs-null, DNA-PKcs kinase-dead and Ku80-null mutants. SCE induction was also abolished in T2609 cluster mutant cells, but was only slightly reduced in the S2056 cluster mutant cells. Since both non-homologous end-joining (NHEJ) and HRR systems utilize initial DNA lesions as a substrate, these results suggest the possibility of a competitive interference phenomenon operating between NHEJ and at least the Rad51C/D components of HRR; the level of interaction between damaged DNA and a particular DNA-PK component may determine the level of interaction of such DNA with a relevant HRR component.
Asunto(s)
Sustitución de Aminoácidos , Aberraciones Cromosómicas/efectos de la radiación , Reparación del ADN por Unión de Extremidades/efectos de la radiación , Proteína Quinasa Activada por ADN/genética , Reparación del ADN por Recombinación/efectos de la radiación , Partículas alfa , Animales , Células CHO , Cricetulus , Rayos gamma , Humanos , Tolerancia a Radiación , Intercambio de Cromátides Hermanas/efectos de la radiaciónRESUMEN
Methyl CpG-binding protein 2 gene (MeCP2) mutations are implicated in Rett syndrome (RTT), one of the common causes of female mental retardation. Two MeCP2 isoforms have been reported: MeCP2_e2 (splicing of all four exons) and MeCP2_e1 (alternative splicing of exons 1, 3, and 4). Their relative expression levels vary among tissues, with MeCP2_e1 being more dominant in adult brain, whereas MeCP2_e2 is expressed more abundantly in placenta, liver, and skeletal muscle. In this study, we performed specific disruption of the MeCP2_e2-defining exon 2 using the Cre-loxP system and examined the consequences of selective loss of MeCP2_e2 function in vivo. We performed behavior evaluation, gene expression analysis, using RT-PCR and real-time quantitative PCR, and histological analysis. We demonstrate that selective deletion of MeCP2_e2 does not result in RTT-associated neurological phenotypes but confers a survival disadvantage to embryos carrying a MeCP2_e2 null allele of maternal origin. In addition, we reveal a specific requirement for MeCP2_e2 function in extraembryonic tissue, where selective loss of MeCP2_e2 results in placenta defects and up-regulation of peg-1, as determined by the parental origin of the mutant allele. Taken together, our findings suggest a novel role for MeCP2 in normal placenta development and illustrate how paternal X chromosome inactivation in extraembryonic tissues confers a survival disadvantage for carriers of a mutant maternal MeCP2_e2 allele. Moreover, our findings provide an explanation for the absence of reports on MeCP2_e2-specific exon 2 mutations in RTT. MeCP2_e2 mutations in humans may result in a phenotype that evades a diagnosis of RTT.