RESUMEN
Short tandem repeat (STR) instability causes transcriptional silencing in several repeat expansion disorders. In fragile X syndrome (FXS), mutation-length expansion of a CGG STR represses FMR1 via local DNA methylation. Here, we find megabase-scale H3K9me3 domains on autosomes and encompassing FMR1 on the X chromosome in FXS patient-derived iPSCs, iPSC-derived neural progenitors, EBV-transformed lymphoblasts, and brain tissue with mutation-length CGG expansion. H3K9me3 domains connect via inter-chromosomal interactions and demarcate severe misfolding of TADs and loops. They harbor long synaptic genes replicating at the end of S phase, replication-stress-induced double-strand breaks, and STRs prone to stepwise somatic instability. CRISPR engineering of the mutation-length CGG to premutation length reverses H3K9me3 on the X chromosome and multiple autosomes, refolds TADs, and restores gene expression. H3K9me3 domains can also arise in normal-length iPSCs created with perturbations linked to genome instability, suggesting their relevance beyond FXS. Our results reveal Mb-scale heterochromatinization and trans interactions among loci susceptible to instability.
Asunto(s)
Síndrome del Cromosoma X Frágil , Humanos , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Expansión de Repetición de Trinucleótido , Metilación de ADN , Mutación , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismoRESUMEN
DNA replication occurs through an intricately regulated series of molecular events and is fundamental for genome stability1,2. At present, it is unknown how the locations of replication origins are determined in the human genome. Here we dissect the role of topologically associating domains (TADs)3-6, subTADs7 and loops8 in the positioning of replication initiation zones (IZs). We stratify TADs and subTADs by the presence of corner-dots indicative of loops and the orientation of CTCF motifs. We find that high-efficiency, early replicating IZs localize to boundaries between adjacent corner-dot TADs anchored by high-density arrays of divergently and convergently oriented CTCF motifs. By contrast, low-efficiency IZs localize to weaker dotless boundaries. Following ablation of cohesin-mediated loop extrusion during G1, high-efficiency IZs become diffuse and delocalized at boundaries with complex CTCF motif orientations. Moreover, G1 knockdown of the cohesin unloading factor WAPL results in gained long-range loops and narrowed localization of IZs at the same boundaries. Finally, targeted deletion or insertion of specific boundaries causes local replication timing shifts consistent with IZ loss or gain, respectively. Our data support a model in which cohesin-mediated loop extrusion and stalling at a subset of genetically encoded TAD and subTAD boundaries is an essential determinant of the locations of replication origins in human S phase.
Asunto(s)
Proteínas de Ciclo Celular , Cromatina , Proteínas Cromosómicas no Histona , Origen de Réplica , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN , Humanos , Origen de Réplica/genética , Fase S , CohesinasRESUMEN
Caspase-2 is unique among all the mammalian caspases in that it is the only caspase that is present constitutively in the cell nucleus, in addition to other cellular compartments. However, the functional significance of this nuclear localization is unknown. Here we show that DNA damage induced by gamma-radiation triggers the phosphorylation of nuclear caspase-2 at the S122 site within its prodomain, leading to its cleavage and activation. This phosphorylation is carried out by the nuclear serine/threonine protein kinase DNA-PKcs and promoted by the p53-inducible death-domain-containing protein PIDD within a large nuclear protein complex consisting of DNA-PKcs, PIDD, and caspase-2, which we have named the DNA-PKcs-PIDDosome. This phosphorylation and the catalytic activity of caspase-2 are involved in the maintenance of a G2/M DNA damage checkpoint and DNA repair mediated by the nonhomologous end-joining (NHEJ) pathway. The DNA-PKcs-PIDDosome thus represents a protein complex that impacts mammalian G2/M DNA damage checkpoint and NHEJ.
Asunto(s)
Proteínas Portadoras/metabolismo , Caspasa 2/metabolismo , Ciclo Celular , Cisteína Endopeptidasas/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Caspasa 2/química , Línea Celular , Cisteína Endopeptidasas/química , Daño del ADN , Proteínas Adaptadoras de Señalización del Receptor del Dominio de Muerte , Fibroblastos/metabolismo , Rayos gamma , Humanos , Ratones , Mitosis , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
Replication fork stability during DNA replication is vital for maintenance of genomic stability and suppression of cancer development in mammals. ATR (ataxia-telangiectasia mutated [ATM] and RAD3-related) is a master regulatory kinase that activates the replication stress response to overcome replication barriers. Although many downstream effectors of ATR have been established, the upstream regulators of ATR and the effect of such regulation on liver cancer remain unclear. The ubiquitin conjugase BRUCE (BIR Repeat containing Ubiquitin-Conjugating Enzyme) is a guardian of chromosome integrity and activator of ATM signaling, which promotes DNA double-strand break repair through homologous recombination. Here we demonstrate the functions for BRUCE in ATR activation in vitro and liver tumor suppression in vivo. BRUCE is recruited to induced DNA damage sites. Depletion of BRUCE inhibited multiple ATR-dependent signaling events during replication stress, including activation of ATR itself, phosphorylation of its downstream targets CHK1 and RPA, and the mono-ubiquitination of FANCD2. Consequently, BRUCE deficiency resulted in stalled DNA replication forks and increased firing of new replication origins. The in vivo impact of BRUCE loss on liver tumorigenesis was determined using the hepatocellular carcinoma model induced by genotoxin diethylnitrosamine. Liver-specific knockout of murine Bruce impaired ATR activation and exacerbated inflammation, fibrosis and hepatocellular carcinoma, which exhibited a trabecular architecture, closely resembling human hepatocellular carcinoma (HCC). In humans, the clinical relevance of BRUCE down-regulation in liver disease was found in hepatitis, cirrhosis, and HCC specimens, and deleterious somatic mutations of the Bruce gene was found in human hepatocellular carcinoma in the Cancer Genome Atlas database. Conclusion: These findings establish a BRUCE-ATR signaling axis in accurate DNA replication and suppression of liver cancer in mice and humans and provides a clinically relevant HCC mouse model.
Asunto(s)
Carcinoma Hepatocelular/genética , Replicación del ADN/genética , Proteínas Inhibidoras de la Apoptosis/genética , Neoplasias Hepáticas/genética , Transducción de Señal/genética , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Carcinogénesis , Carcinoma Hepatocelular/patología , Reparación del ADN/genética , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Inestabilidad Genómica , Humanos , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Noqueados , Distribución Aleatoria , Sensibilidad y Especificidad , Proteínas Supresoras de Tumor/genéticaRESUMEN
The DNA damage response (DDR) is crucial for genomic integrity. BRIT1 (breast cancer susceptibility gene C terminus-repeat inhibitor of human telomerase repeat transcriptase expression), a tumor suppressor and early DDR factor, is recruited to DNA double-strand breaks (DSBs) by phosphorylated H2A histone family, member X (γ-H2AX), where it promotes chromatin relaxation by recruiting the switch/sucrose nonfermentable (SWI-SNF) chromatin remodeler to facilitate DDR. However, regulation of BRIT1 recruitment is not fully understood. The baculovirus IAP repeat (BIR)-containing ubiquitin-conjugating enzyme (BRUCE) is an inhibitor of apoptosis protein (IAP). Here, we report a non-IAP function of BRUCE in the regulation of the BRIT1-SWI-SNF DSB-response pathway and genomic stability. We demonstrate that BRIT1 is K63 ubiquitinated in unstimulated cells and that deubiquitination of BRIT1 is a prerequisite for its recruitment to DSB sites by γ-H2AX. We show mechanistically that BRUCE acts as a scaffold, bridging the ubiquitin-specific peptidase 8 (USP8) and BRIT1 in a complex to coordinate USP8-catalyzed deubiquitination of BRIT1. Loss of BRUCE or USP8 impairs BRIT1 deubiquitination, BRIT1 binding with γ-H2AX, the formation of BRIT1 DNA damage foci, and chromatin relaxation. Moreover, BRUCE-depleted cells display reduced homologous recombination repair, and BRUCE-mutant mice exhibit repair defects and genomic instability. These findings identify BRUCE and USP8 as two hitherto uncharacterized critical DDR regulators and uncover a deubiquitination regulation of BRIT1 assembly at damaged chromatin for efficient DDR and genomic stability.
Asunto(s)
Roturas del ADN de Doble Cadena , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación , Animales , Proteínas de Ciclo Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Cromosomas de los Mamíferos/metabolismo , Proteínas del Citoesqueleto , Reparación del ADN , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Inestabilidad Genómica , Células HEK293 , Histonas/metabolismo , Recombinación Homóloga/genética , Humanos , Lisina/metabolismo , Ratones , Complejos Multiproteicos/metabolismo , Poliubiquitina/metabolismo , Transporte de ProteínasRESUMEN
The tumor suppressor gene, BTG2 has been down-regulated in prostate cancer and the ectopic expression of this gene has been shown to inhibit prostate cancer cell growth. Sequence analysis revealed that the BTG2 protein contains two leucine-rich motifs ((20)LxxLL(24) and (92)LxxLL(96)), which are usually found in nuclear receptor co-factors. Based on this, we postulated that there will be an association between BTG2 and AR. In this study, we discovered that BTG2 directly bound to the androgen receptor (AR) in the absence of 5α-dihydrotestosterone (DHT), and in the presence of the androgen, this interaction was increased. BTG2 bearing the mutant (20)LxxLL(24) motif bound to AR equally efficient as the wild-type BTG2, while BTG2 bearing the mutant (92)LxxLL(96) motif failed to interact with AR. Functional studies indicated that ectopic expression of BTG2 caused a significant inhibition of AR-mediated transcriptional activity and a decreased growth of prostate cancer cells. Androgen-induced promoter activation and expression of prostate-specific antigen (PSA) are significantly attenuated by BTG2. The intact (92)LxxLL(96) motif is required for these activities. These findings, for the first time, demonstrate that BTG2 complexes with AR via an LxxLL-dependent mechanism and may play a role in prostate cancer via modulating the AR signaling pathway.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas Inmediatas-Precoces/metabolismo , Leucina Zippers , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Receptores Androgénicos/metabolismo , Proteínas Represoras/metabolismo , Proteínas Supresoras de Tumor/metabolismo , 5-alfa-Dihidroprogesterona/metabolismo , Línea Celular Tumoral , Proliferación Celular , Humanos , Proteínas Inmediatas-Precoces/genética , Masculino , Mutación , Neoplasias de la Próstata/metabolismo , Receptores Androgénicos/genética , Proteínas Represoras/genética , Transcripción Genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia , Metabolismo Energético , Proteínas Inhibidoras de la Apoptosis/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Osteosarcoma/patología , Neoplasias Ováricas/patología , Proteínas Quinasas Activadas por AMP/genética , Autofagosomas , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Neoplasias Óseas/genética , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Supervivencia Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Lisosomas/metabolismo , Osteosarcoma/genética , Osteosarcoma/metabolismo , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Fosforilación , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Polyamines are implicated in regulating various developmental processes in plants, but their exact roles and how they govern these processes still remain elusive. We report here an Arabidopsis bushy and dwarf mutant, bud2, which results from the complete deletion of one member of the small gene family that encodes S-adenosylmethionine decarboxylases (SAMDCs) necessary for the formation of the indispensable intermediate in the polyamine biosynthetic pathway. The bud2 plant has enlarged vascular systems in inflorescences, roots, and petioles, and an altered homeostasis of polyamines. The double mutant of bud2 and samdc1, a knockdown mutant of another SAMDC member, is embryo lethal, demonstrating that SAMDCs are essential for plant embryogenesis. Our results suggest that polyamines are required for the normal growth and development of higher plants.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Adenosilmetionina Descarboxilasa/fisiología , Secuencia de Aminoácidos , Arabidopsis/embriología , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/fisiología , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Mutación , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Plantas Modificadas Genéticamente , Semillas/genética , Semillas/crecimiento & desarrollo , Homología de Secuencia de AminoácidoRESUMEN
BRUCE is implicated in the regulation of DNA double-strand break response to preserve genome stability. It acts as a scaffold to tether USP8 and BRIT1, together they form a nuclear BRUCE-USP8-BRIT1 complex, where BRUCE holds K63-ubiquitinated BRIT1 from access to DSB in unstressed cells. Following DSB induction, BRUCE promotes USP8 mediated deubiquitination of BRIT1, a prerequisite for BRIT1 to be released from the complex and recruited to DSB by binding to γ-H2AX. BRUCE contains UBC and BIR domains, but neither is required for the scaffolding function of BRUCE mentioned above. Therefore, it remains to be determined whether they are required for BRUCE in DSB response. Here we show that the UBC domain, not the BIR domain, is required for BRUCE to promote DNA repair at a step post the formation of BRUCE-USP8-BRIT1 complex. Mutation or deletion of the BRUCE UBC domain did not disrupt the BRUCE-USP8-BRIT1 complex, but impaired deubiquitination and consequent recruitment of BRIT1 to DSB. This leads to impaired chromatin relaxation, decreased accumulation of MDC1, NBS1, pATM and RAD51 at DSB, and compromised homologous recombination repair of DNA DSB. These results demonstrate that in addition to the scaffolding function in complex formation, BRUCE has an E3 ligase function to promote BRIT1 deubiquitination by USP8 leading to accumulation of BRIT1 at DNA double-strand break. These data support a crucial role for BRUCE UBC activity in the early stage of DSB response.
Asunto(s)
Reparación del ADN , Endopeptidasas/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Proteínas Inhibidoras de la Apoptosis/química , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Proteínas de Ciclo Celular , Línea Celular , Cromatina/fisiología , Proteínas del Citoesqueleto , Roturas del ADN de Doble Cadena , Células HEK293 , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Mutación , Estructura Terciaria de Proteína , Transducción de Señal , UbiquitinaciónRESUMEN
BACKGROUND AND OBJECTIVES: Management of patients with breast cancer often fails because of inherent or acquired resistance to chemotherapy. BRUCE (BIR repeat containing ubiquitin-conjugating enzyme) is a member of the inhibitor of apoptosis protein (IAP) family. It has various cellular functions including suppression of apoptosis and promotion of cytokinesis. Furthermore, it pays a critical role in promotion of DNA damage repair and preservation of genome stability, a new function recently reported by our group. Although BRUCE is expressed in breast cancer cell lines, its expression in human primary breast tumors and its contribution to chemoresistance in breast cancers has not been explored. Chemotherapeutic drugs are used in the treatment of breast cancer patients. However, they are not effective to all patients and patients often develop resistance. Consequently we explored if BRUCE protein level, as judged by immunohistochemistry (IHC), is higher in primary breast tumors than normal breast tissue. We also examined if depletion of BRUCE, using a lentiviral shRNA approach, enhances cell sensitivity to multiple chemotherapeutic agents, including cisplatin, an agent that induces DNA damage by generating DNA cross-links, and taxol, a microtubule stabilizer and mitotic inhibitor. The reason for including these two chemotherapeutic agents in this study is that they hit two essential cellular processes of DNA repair and cytokinesis in which BRUCE plays critical roles. RESULTS AND METHODS: IHC analysis of BRUCE revealed significantly higher levels of BRUCE in primary breast tumors than normal breast tissue. Knockdown of BRUCE protein expression by lentiviral shRNA resulted in increased sensitivity to cisplatin in the resistant breast cancer MDB-MD-231 cell line. Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone. CONCLUSIONS: Our data suggest that elevated protein levels of BRUCE in breast tumors may contribute to chemoresistance in breast cancer patients. In support of this suggestion, our data demonstrate that a reduction in BRUCE expression in breast cancer cell lines increases the toxicity of several chemotherapeutic agents. In all likelihood, the contribution of increased BRUCE levels to chemoresistance are likely due to its roles in suppression of apoptosis, promotion of cytokinesis and facilitation of DNA damage repair. These observations suggest that therapeutic suppression of BRUCE could improve chemosensitivity in chemo-resistant breast cancer patients. Therefore, future development of effective inhibitors of BRUCE could benefit patients with high BRUCE expression and chemoresistance.
RESUMEN
Our previous studies have shown that high-mobility group box 1 (HMGB1) could physically associate with the retinoblastoma (RB) protein via an LXCXE (leucine-X-cysteine-X-glutamic; X=any amino acid) motif. An identical LXCXE motif is present in the HMGB1-3 protein sequences, whereas a near-consensus LXCXD (leucine-X-cysteine-X-asparagine; X=any amino acid) motif is found in the HMGB4 protein. In this study, we have demonstrated that like HMGB1, HMGB2-3 also associated with the RB in vitro and in vivo, as evidenced by glutathione-s-transferase capture and immunoprecipitation-Western blot assays. A point mutation of the LXCXE or LXCXD motif led to disruption of RB:HMGB1-4 interactions. Enforced expression of HMGB1-3 or HMGB4 by adenoviral-vector-mediated gene transfer resulted in significant inhibition of breast cancer cell proliferation through an LXCXE- or LXCXD-dependent mechanism and an increased radiosensitivity through an LXCXE- or LXCXD-independent mechanism. These results suggest an important role of the LXCXE/D motif in RB:HMGB1-4 association and modulation of cancer cell growth, but not radiosensitivity.
Asunto(s)
Neoplasias de la Mama/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Proteína de Retinoblastoma/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/radioterapia , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Femenino , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Tolerancia a Radiación , Retinoblastoma/genética , Retinoblastoma/metabolismo , Retinoblastoma/patología , Proteína de Retinoblastoma/genética , TransfecciónRESUMEN
The ratio of auxin and cytokinin plays a crucial role in regulating aerial architecture by promoting or repressing axillary bud outgrowth. We have previously identified an Arabidopsis mutant bud2 that displays altered root and shoot architecture, which results from the loss-of-function of S-adenosylmethionine decarboxylase 4 (SAMDC4). In this study, we demonstrate that BUD2 could be induced by auxin, and the induction is dependent on auxin signaling. The mutation of BUD2 results in hyposensitivity to auxin and hypersensitivity to cytokinin, which is confirmed by callus induction assays. Our study suggests that polyamines may play their roles in regulating the plant architecture through affecting the homeostasis of cytokinins and sensitivities to auxin and cytokinin.
Asunto(s)
Adenosilmetionina Descarboxilasa/genética , Proteínas de Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/genética , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Mutación , Adenosilmetionina Descarboxilasa/metabolismo , Adenilil Ciclasas , Proteínas de Arabidopsis/metabolismo , Raíces de Plantas/crecimiento & desarrolloRESUMEN
AIM: To investigate the effect of the Tob1 gene, a member of the Transducing Molecule of ErbB2/B-cell Translocation Ggene (TOB/BTG) family, by using the adenovirus-mediated expression of Tob1 on radiosensitivity in a human breast cancer cell line MDA-MB-231. METHODS: Cell survival was determined by clonogenic assay. Apoptosis was evaluated by DNA fragmentation gel electrophoresis and terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Protein expression was analyzed by Western blot assay and DNA repair was measured by a host cell reactivation assay. RESULTS: We demonstrated that pre-irradiation treatment with Ad5-Tob1 significantly increased radiosensitivity, accompanying the increased induction of apoptosis and the repression of DNA damage repair. Furthermore, Ad5-Tob1-mediated radiosensitivity correlates with the upregulation of the pro-apoptotic protein Bax and the downregulation of several DNA double strand break repair proteins, including DNA-dependent protein kinases, Ku70 and Ku80, and X-ray-sensitive complementation group 4. CONCLUSION: Tob1, as a new radiosensitizer, is a new target in the radiotherapy of breast cancer via increasing apoptosis and suppressing DNA repair.
Asunto(s)
Adenoviridae/genética , Apoptosis/efectos de la radiación , Péptidos y Proteínas de Señalización Intracelular/fisiología , Radiación Ionizante , Proteínas Supresoras de Tumor/fisiología , Antígenos Nucleares/metabolismo , Apoptosis/genética , Apoptosis/fisiología , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Radioisótopos de Cesio , Reparación del ADN/efectos de la radiación , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Vectores Genéticos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Autoantígeno Ku , Proteínas Nucleares/metabolismo , Fármacos Sensibilizantes a Radiaciones/metabolismo , Transfección , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
AIM: To investigate the anticancer activity of dihydroartemisinin (DHA), a derivative of antimalaria drug artemisinin in a panel of human ovarian cancer cell lines. METHODS: Cell growth was determined by the MTT viability assay. Apoptosis and cell cycle progression were evaluated by a DNA fragmentation gel electro-phoresis, flow cytometry assay, and TUNEL assay; protein and mRNA expression were analyzed by Western blotting and RT-PCR assay. RESULTS: Artemisinin and its derivatives, including artesunate, arteether, artemether, arteannuin, and DHA, exhibit anticancer growth activities in human ovarian cancer cells. Among them, DHA is the most effective in inhibiting cell growth. Ovarian cancer cell lines are more sensitive (5-10-fold) to DHA treatment compared to normal ovarian cell lines. DHA at micromolar dose levels exhibits a dose- and time-dependent cytotoxicity in ovarian cancer cell lines. Furthermore, DHA induced apoptosis and G2 cell cycle arrest, accompanied by a decrease of Bcl-xL and Bcl-2 and an increase of Bax and Bad. CONCLUSION: The promising results show for the first time that DHA inhibits the growth of human ovarian cancer cells. The selective inhibition of ovarian cancer cell growth, apoptosis induction, and G2 arrest provide in vitro evidence for further studies of DHA as a possible anticancer drug in the clinical treatment of ovarian cancer.