RESUMEN
Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double-stranded breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Ablating phosphorylation at T4102 attenuates DNA-PKcs kinase activity and this destabilizes the interaction between DNA-PKcs and the Ku-DNA complex, resulting in decreased assembly and stabilization of the NHEJ machinery at DSBs. Phosphorylation at T4102 promotes NHEJ, radioresistance, and increases genomic stability following DSB induction. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PKcs.
Asunto(s)
Ataxia Telangiectasia , Proteína Quinasa Activada por ADN , Humanos , Proteína Quinasa Activada por ADN/genética , Reparación del ADN , Treonina/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Reparación del ADN por Unión de Extremidades , ADN/genéticaRESUMEN
Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activation of multiple LPA G protein-coupled receptors. We previously reported that LPA suppresses oxidative stress in premature aging Hutchinson-Gilford progeria syndrome (HGPS) patient fibroblasts via its type 3 receptor (LPA3). Mitochondria have been suggested to be the primary origin of oxidative stress via the overproduction of reactive oxygen species (ROS). Mitochondria are responsible for producing ATP through oxidative phosphorylation (OXPHOS) and have a calcium buffering capacity for the cell. Defects in mitochondria will lead to declined antioxidant capacity and cell apoptosis. Therefore, we aim to demonstrate the regulatory role of LPA3 in mitochondrial homeostasis. siRNA-mediated depletion of LPA3 leads to the depolarization of mitochondrial potential (ΔΨm) and cellular ROS accumulation. In addition, the depletion of LPA3 enhances cisplatin-induced cytochrome C releasing. This indicates that LPA3 is essential to suppress the mitochondrial apoptosis pathway. LPA3 is also shown to improve mitochondrial ADP-ATP exchange by enhancing the protein level of ANT2. On the other hand, LPA3 regulates calcium uptake from the ER to mitochondria via the IP3R1-VDAC1 channel. Moreover, activation of LPA3 by selective agonist OMPT rescues mitochondrial homeostasis of H2O2-induced oxidative stress cells and HGPS patient fibroblasts by improving mitochondrial ΔΨm and OXPHOS. In summary, our findings imply that LPA3 acts as the gatekeeper for mitochondrial healthiness to maintain cell youth. Furthermore, LPA3 can be a promising therapeutic target to prevent mitochondrial oxidative stress in aging and HGPS.
RESUMEN
The heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), telomeric repeat-containing RNA (TERRA), and protection of telomeres 1 (POT1) have been reported to orchestrate to displace replication protein A (RPA) from telomeric overhangs, ensuring orderly telomere replication and capping. Our previous studies further demonstrated that DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-dependent hnRNPA1 phosphorylation plays a crucial role in the promotion of hnRNPA1 binding to telomeric overhangs and RPA displacement during G2-M phases. However, it is unclear that how the subsequent exchange between hnRNPA1 and POT1 is orchestrated. Here we report that the protein phosphatase 2A (PP2A) depends on its scaffold subunit, which is called PPP2R1A, to interact with and dephosphorylate hnRNPA1 in the late M phase. Furthermore, PP2A-mediated hnRNPA1 dephosphorylation and TERRA accumulation act in concert to promote the hnRNPA1-to-POT1 switch on telomeric single-stranded DNA. Consequently, defective PPP2R1A results in ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage response at telomeres as well as induction of fragile telomeres. Combined inhibition of ATR and PP2A induces entry into a catastrophic mitosis and leads to synthetic lethality of tumor cells. In addition, PPP2R1A levels correlate with clinical stages and prognosis of multiple types of cancers. Taken together, our results indicate that PP2A is critical for telomere maintenance. IMPLICATIONS: This study demonstrates that the PP2A-dependent hnRNPA1 dephosphorylation and TERRA accumulation facilitates the formation of the protective capping structure of newly replicated telomeres, thus exerting essential oncogenic role in tumorigenesis.
Asunto(s)
Proteína Fosfatasa 2 , Proteínas de Unión a Telómeros , Proteínas de Unión al ADN , Ribonucleoproteína Nuclear Heterogénea A1/genética , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Humanos , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , Telómero/genética , Telómero/metabolismo , Proteínas de Unión a Telómeros/genética , Factores de TranscripciónRESUMEN
Chromosomal instability (CIN) is a driving force for cancer development. The most common causes of CIN include the dysregulation of the spindle assembly checkpoint (SAC), which is a surveillance mechanism that prevents premature chromosome separation during mitosis by targeting anaphase-promoting complex/cyclosome (APC/C). DAB2IP is frequently silenced in advanced prostate cancer (PCa) and is associated with aggressive phenotypes of PCa. Our previous study showed that DAB2IP activates PLK1 and functions in mitotic regulation. Here, we report the novel mitotic phosphorylation of DAB2IP by Cdks, which mediates DAB2IP's interaction with PLK1 and the activation of the PLK1-Mps1 pathway. DAB2IP interacts with Cdc20 in a phosphorylation-independent manner. However, the phosphorylation of DAB2IP inhibits the ubiquitylation of Cdc20 in response to SAC, and blocks the premature release of the APC/C-MCC. The PLK1-Mps1 pathway plays an important role in mitotic checkpoint complex (MCC) assembly. It is likely that DAB2IP acts as a scaffold to aid PLK1-Mps1 in targeting Cdc20. Depletion or loss of the Cdks-mediated phosphorylation of DAB2IP destabilizes the MCC, impairs the SAC, and increases chromosome missegregation and subsequent CIN, thus contributing to tumorigenesis. Collectively, these results demonstrate the mechanism of DAB2IP in SAC regulation and provide a rationale for targeting the SAC to cause lethal CIN against DAB2IP-deficient aggressive PCa, which exhibits a weak SAC.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Inestabilidad Cromosómica/genética , Mitosis/genética , Oncogenes/genética , Huso Acromático/metabolismo , Humanos , Fosforilación , Transducción de Señal , TransfecciónRESUMEN
RNA polymerase II (Pol II)-dependent transcription in stimulus-inducible genes requires topoisomerase IIß (TOP2B)-mediated DNA strand break and the activation of DNA damage response signalling in humans. Here, we report a novel function of the breast cancer 1 (BRCA1)-BRCA1-associated ring domain 1 (BARD1) complex in this process. We found that BRCA1 is phosphorylated at S1524 by the kinases ataxia-telangiectasia mutated and ATR during gene activation, and that this event is important for productive transcription. Our biochemical and genomic analyses showed that the BRCA1-BARD1 complex interacts with TOP2B in the EGR1 transcription start site and in a large number of protein-coding genes. Intriguingly, the BRCA1-BARD1 complex ubiquitinates TOP2B, which stabilizes TOP2B binding to DNA while BRCA1 phosphorylation at S1524 controls the TOP2B ubiquitination by the complex. Together, these findings suggest the novel function of the BRCA1-BARD1 complex in the regulation of TOP2B and Pol II-mediated gene expression.
Asunto(s)
Proteína BRCA1/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteína BRCA1/química , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Mutación , Fosforilación , Sitio de Iniciación de la Transcripción , Transcripción Genética , UbiquitinaciónRESUMEN
PURPOSE: Harnessing the immune-stimulatory effects of radiation by combining it with immunotherapy is a promising new treatment strategy. However, more studies characterizing immunotherapy and radiation dose scheduling for the optimal therapeutic effect is essential for designing clinical trials. METHODS AND MATERIALS: A new ablative radiation dosing scheme, personalized ultrafractionated stereotactic adaptive radiation therapy (PULSAR), was tested in combination with α-PD-L1 therapy in immune-activated and resistant syngeneic immunocompetent mouse models of cancer. Specifically, tumor growth curves comparing immunotherapy and radiation therapy dose sequencing were evaluated in immunologically cold and hot tumor models. The response relative to cytotoxic killer T cells was evaluated using an α-CD8 depleting antibody, and immunologic memory was tested by tumor rechallenge of cured mice. RESULTS: We report that both radiation and immunotherapy sequencing, as well as radiation therapy fraction spacing, affect the combination treatment response. Better tumor control was achieved by giving α-PD-L1 therapy during or after radiation, and spacing fractions 10 days apart (PULSAR) achieved better tumor control than traditional daily fractions. We showed that CD8+ depleting antibody abrogated tumor control in the PULSAR combination treatment, and certain treatment schedules induced immunologic memory. CONCLUSIONS: These results illustrate that radiation therapy dosing and scheduling affect tumor control, in combination with checkpoint blockade therapies. PULSAR-style radiation dosing is more complementary in combination with single-agent immunotherapy than traditional daily fractions in this preclinical model. Preclinical investigation could prove helpful in designing clinical trials investigating combination therapy.
Asunto(s)
Carcinoma Pulmonar de Lewis/terapia , Neoplasias del Colon/terapia , Fraccionamiento de la Dosis de Radiación , Inhibidores de Puntos de Control Inmunológico/farmacología , Medicina de Precisión/métodos , Radioinmunoterapia/métodos , Radiocirugia/métodos , Animales , Antígeno B7-H1 , Carcinoma Pulmonar de Lewis/inmunología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Femenino , Memoria Inmunológica , Ratones , Ratones Endogámicos C57BL , Dosificación Radioterapéutica , Distribución Aleatoria , Linfocitos T Citotóxicos , Resultado del TratamientoRESUMEN
Combining targeted therapeutic agents is an attractive cancer treatment strategy associated with high efficacy and low toxicity. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is an essential factor in DNA damage repair. Studies from us and others have revealed that DNA-PKcs also plays an important role in normal mitosis progression. Histone deacetylase (HDACs) inhibitors commonly lead to mitotic aberration and have been approved for treating various cancers in the clinic. We showed that DNA-PKcs depletion or kinase activity inhibition increases cancer cells' sensitivity to HDACs inhibitors in vitro and in vivo. DNA-PKcs deficiency significantly enhances HDACs inhibitors (HDACi)-induced mitotic arrest and is followed by apoptotic cell death. Mechanistically, we found that DNA-PKcs binds to HDAC6 and facilitates its acetylase activity. HDACi is more likely to impair HDAC6-induced deacetylation of HSP90 and abrogate HSP90's chaperone function on Aurora A, a critical mitotic kinase that regulates centrosome separation and mitotic spindle assembly in DNA-PKcs-deficient cells. Our current work indicates crosstalk between DNA-PKcs and HDACs signaling pathways, and highlights that the combined targeting of DNA-PKcs and HDACs can be used in cancer therapy. Abbreviations: DNA-PKcs, DNA-dependent protein kinase catalytic subunit, HDACs, Histone deacetylases, DSBs, DNA double-strand breaks, ATM, ataxia telangiectasia mutated, ATR, ATM-Rad3-related.
Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Histona Desacetilasa 6/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/metabolismo , División del Núcleo Celular/genética , División del Núcleo Celular/fisiología , Daño del ADN/genética , Reparación del ADN/genética , Histona Desacetilasa 6/genética , Humanos , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Hematopoiesis, the complex developmental process that forms blood components and replenishes the blood system, involves multiple intracellular and extracellular mechanisms. We previously demonstrated that lysophosphatidic acid (LPA), a lipid growth factor, has opposing regulatory effects on erythrocyte differentiation through activation of LPA receptors 2 and 3; yet the mechanisms underlying this process remain unclear. In this study, LPA2 is observed that highly expressed in common myeloid progenitors (CMP) in murine myeloid cells, whereas the expression of LPA3 displaces in megakaryocyte-erythroid progenitors (MEP) of later stage of myeloid differentiation. Therefore, we hypothesized that the switching expression of LPA2 and LPA3 determine the hematic homeostasis of mammalian megakaryocytic-erythroid lineage. In vitro colony-forming unit assays of murine progenitors reveal that LPA2 agonist GRI reduces the erythroblast differentiation potential of CMP. In contrast, LPA3 agonist OMPT increases the production of erythrocytes from megakaryocyte-erythrocyte progenitor cells (MEP). In addition, treatment with GRI reduces the erythroid, CMP, and MEP populations in mice, indicating that LPA2 predominantly inhibits myeloid differentiation at an early stage. In contrast, activation of LPA3 increases the production of terminally differentiated erythroid cells through activation of erythropoietic transcriptional factor. We also demonstrate that the LPA3 signaling is essential for restoration of phenylhydrazine (PHZ)-induced acute hemolytic anemia in mice and correlates to erythropoiesis impairment of Hutchinson-Gilford progeria Symptom (HGPS) premature aging expressed K562 model. Our results reveal the distinct roles of LPA2 and LPA3 at different stages of hematopoiesis in vivo, providing potentiated therapeutic strategies of anemia treatment.
Asunto(s)
Anemia Hemolítica/genética , Células Eritroides/metabolismo , Eritropoyesis/genética , Células Mieloides/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Células Madre/metabolismo , Anemia Hemolítica/inducido químicamente , Anemia Hemolítica/tratamiento farmacológico , Anemia Hemolítica/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/genética , Modelos Animales de Enfermedad , Células Eritroides/citología , Células Eritroides/efectos de los fármacos , Eritropoyesis/efectos de los fármacos , Regulación de la Expresión Génica , Humanos , Isoquinolinas/farmacología , Células K562 , Lisofosfolípidos/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Células Mieloides/citología , Células Mieloides/efectos de los fármacos , Organotiofosfatos/farmacología , Fenilhidrazinas/administración & dosificación , Ácidos Fosfatidicos/farmacología , Receptores del Ácido Lisofosfatídico/agonistas , Receptores del Ácido Lisofosfatídico/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacosRESUMEN
In mammalian cells, extracellular vesicles (EVs) derived from the endosomal system carry many different kinds of bioactive molecule to deliver to recipient cells in a paracrine or endocrine manner. EVs can mediate local and systemic intercellular communications, including reeducating stromal cells, remodeling the architecture of the tumor microenvironment, modulating cancer metabolism and metastases, or even conferring drug resistance. Because the molecular and functional characteristics of prostate cancer (PCa) evolve over time, the bioactive molecule profiles/signatures of tumor-derived EVs (TDEs) reflect the real-time status of cancer cells. TDEs appear to be valuable diagnostic and prognostic biomarkers as well as potential therapeutic vehicles, suggesting their essential role in precision medicine of disease management. We summarized critical aspects of TDEs in PCa and discussed their potential clinical applications.
Asunto(s)
Vesículas Extracelulares/metabolismo , Neoplasias de la Próstata/metabolismo , Biomarcadores de Tumor/metabolismo , Humanos , Masculino , PronósticoRESUMEN
Astronauts on interplanetary missions - such as to Mars - will be exposed to space radiation, a spectrum of highly-charged, fast-moving particles that includes 56Fe and 28Si. Earth-based preclinical studies show space radiation decreases rodent performance in low- and some high-level cognitive tasks. Given astronaut use of touchscreen platforms during training and space flight and given the ability of rodent touchscreen tasks to assess functional integrity of brain circuits and multiple cognitive domains in a non-aversive way, here we exposed 6-month-old C57BL/6J male mice to whole-body space radiation and subsequently assessed them on a touchscreen battery. Relative to Sham treatment, 56Fe irradiation did not overtly change performance on tasks of visual discrimination, reversal learning, rule-based, or object-spatial paired associates learning, suggesting preserved functional integrity of supporting brain circuits. Surprisingly, 56Fe irradiation improved performance on a dentate gyrus-reliant pattern separation task; irradiated mice learned faster and were more accurate than controls. Improved pattern separation performance did not appear to be touchscreen-, radiation particle-, or neurogenesis-dependent, as 56Fe and 28Si irradiation led to faster context discrimination in a non-touchscreen task and 56Fe decreased new dentate gyrus neurons relative to Sham. These data urge revisitation of the broadly-held view that space radiation is detrimental to cognition.
Asunto(s)
Cognición/efectos de la radiación , Radiación Cósmica , Giro Dentado/efectos de la radiación , Aprendizaje por Asociación de Pares/efectos de la radiación , Reconocimiento Visual de Modelos/efectos de la radiación , Aprendizaje Inverso/efectos de la radiación , Animales , Astronautas , Ciencias Bioconductuales , Cognición/fisiología , Giro Dentado/fisiología , Isótopos de Hierro , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/fisiología , Neuronas/efectos de la radiación , Aprendizaje por Asociación de Pares/fisiología , Reconocimiento Visual de Modelos/fisiología , Aprendizaje Inverso/fisiología , Vuelo Espacial , Irradiación Corporal TotalRESUMEN
This review focuses on DNA-dependent protein kinase (DNA-PK), which is the key regulator of canonical non-homologous end-joining (NHEJ), the predominant mechanism of DNA double-strand break (DSB) repair in mammals. DNA-PK consists of the DNA-binding Ku70/80 heterodimer and the catalytic subunit DNA-PKcs. They assemble at DNA ends, forming the active DNA-PK complex, which initiates NHEJ-mediated DSB repair. Paradoxically, both Ku and DNA-PKcs are associated with telomeres, and they play crucial roles in protecting the telomere against fusions. Herein, we discuss possible mechanisms and contributions of Ku and DNA-PKcs in telomere regulation.
Asunto(s)
Proteína Quinasa Activada por ADN/metabolismo , Ribonucleoproteína Nuclear Heterogénea A1/metabolismo , Telomerasa/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Animales , Reparación del ADN por Unión de Extremidades/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteína Quinasa Activada por ADN/química , Proteína Quinasa Activada por ADN/genética , Humanos , Autoantígeno Ku/metabolismo , Complejo Shelterina , Telómero/genéticaRESUMEN
RUVBL1 and RUVBL2 (collectively RUVBL1/2) are essential AAA+ ATPases that function as co-chaperones and have been implicated in cancer. Here we investigated the molecular and phenotypic role of RUVBL1/2 ATPase activity in non-small cell lung cancer (NSCLC). We find that RUVBL1/2 are overexpressed in NSCLC patient tumors, with high expression associated with poor survival. Utilizing a specific inhibitor of RUVBL1/2 ATPase activity, we show that RUVBL1/2 ATPase activity is necessary for the maturation or dissociation of the PAQosome, a large RUVBL1/2-dependent multiprotein complex. We also show that RUVBL1/2 have roles in DNA replication, as inhibition of its ATPase activity can cause S-phase arrest, which culminates in cancer cell death via replication catastrophe. While in vivo pharmacological inhibition of RUVBL1/2 results in modest antitumor activity, it synergizes with radiation in NSCLC, but not normal cells, an attractive property for future preclinical development.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Portadoras/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Neoplasias Pulmonares/metabolismo , Complejos Multiproteicos/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/antagonistas & inhibidores , ATPasas Asociadas con Actividades Celulares Diversas/genética , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/genética , Replicación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/tratamiento farmacológico , Estructura Molecular , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/genética , Tolerancia a RadiaciónRESUMEN
Vanillin is a natural compound endowed with antioxidant and anti-mutagenic properties. We previously identified the vanillin derivative VND3207 with strong radio-protective and antioxidant effects and found that VND3207 confers survival benefit and protection against radiation-induced intestinal injury (RIII) in mice. We also observed that VND3207 treatment enhanced the expression level of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) in human lymphoblastoid cells with or without γ-irradiation. DNA-PKcs is a critical component of DNA double strand break repair pathway and also regulates mitotic progression by stabilizing spindle formation and preventing mitotic catastrophe in response to DNA damage. In the present study, we found that VND3207 protected intestinal epithelial cells in vitro against ionizing radiation by promoting cell proliferation and inhibiting cell apoptosis. In addition, VND3207 promoted DNA-PKcs activity by increasing autophosphorylation at S2056 site. Consistent with this, VND3207 significantly decreased the number of γH2AX foci and mitotic catastrophe after radiation. DNA-PKcs deficiency abolished these VND3207 radio-protective effects, indicating that DNA-PKcs activation is essential for VND3207 activity. In conclusion, VND3207 promoted intestinal repair following radiation injury by regulating the DNA-PKcs pathway.
Asunto(s)
Benzaldehídos/farmacología , Proteína Quinasa Dependiente de GMP Cíclico Tipo I/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Mucosa Intestinal/efectos de los fármacos , Traumatismos Experimentales por Radiación/prevención & control , Protectores contra Radiación/farmacología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Proteína Quinasa Activada por ADN/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Células Epiteliales/efectos de la radiación , Rayos gamma/efectos adversos , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/patología , Mucosa Intestinal/efectos de la radiación , Mutación con Pérdida de Función , Masculino , Ratones , Fosforilación/efectos de los fármacos , Traumatismos Experimentales por Radiación/patología , Protectores contra Radiación/uso terapéuticoRESUMEN
Hutchinson-Gilford progeria syndrome (HGPS) is a rare laminopathy that produces a mutant form of prelamin A, known as Progerin, resulting in premature aging. HGPS cells show morphological abnormalities of the nuclear membrane, reduced cell proliferation rates, accumulation of reactive oxygen species (ROS), and expression of senescence markers. Lysophosphatidic acid (LPA) is a growth factor-like lipid mediator that regulates various physiological functions via activating multiple LPA G protein-coupled receptors. Here, we study the roles of LPA and LPA receptors in premature aging. We report that the protein level of LPA3 was highly downregulated through internalization and the lysosomal degradation pathway in Progerin-transfected HEK293 cells. By treating Progerin HEK293 cells with an LPA3 agonist (OMPT, 1-Oleoyl-2-O-methyl-rac-glycerophosphothionate) and performing shRNA knockdown of the Lpa3r transcript in these cells, we showed that LPA3 activation increased expression levels of antioxidant enzymes, consequently inhibiting ROS accumulation and ameliorating cell senescence. LPA3 was shown to be downregulated in HGPS patient fibroblasts through the lysosomal pathway, and it was shown to be crucial for ameliorating ROS accumulation and cell senescence in fibroblasts. Moreover, in a zebrafish model, LPA3 deficiency was sufficient to cause premature aging phenotypes in multiple organs, as well as a shorter lifespan. Taken together, these findings identify the decline of LPA3 as a key contributor to the premature aging phenotypes of HGPS cells and zebrafish.
Asunto(s)
Progeria/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Animales , Senescencia Celular/fisiología , Técnicas de Silenciamiento del Gen , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lamina Tipo A/biosíntesis , Organotiofosfatos/farmacología , Estrés Oxidativo , Ácidos Fosfatidicos/farmacología , Progeria/patología , Especies Reactivas de Oxígeno/metabolismo , Pez CebraRESUMEN
The intestine is a highly radiosensitive tissue that is susceptible to structural and functional damage due to systemic as well as localized radiation exposure. Unfortunately, no effective prophylactic or therapeutic agents are available at present to manage radiation-induced intestinal injuries. We observed that the vanillin derivative VND3207 improved the survival of lethally irradiated mice by promoting intestinal regeneration and increasing the number of surviving crypts. Pre-treatment with VND3207 significantly increased the number of Lgr5+ intestinal stem cells (ISCs) and their daughter cells, the transient Ki67+ proliferating cells. Mechanistically, VND3207 decreased oxidative DNA damage and lipid peroxidation and maintained endogenous antioxidant status by increasing the level of superoxide dismutase and total antioxidant capacity. In addition, VND3207 maintained appropriate levels of activated p53 that triggered cell cycle arrest but were not sufficient to induce NOXA-mediated apoptosis, thus ensuring DNA damage repair in the irradiated small intestinal crypt cells. Furthermore, VND3207 treatment restores the intestinal bacterial flora structures altered by TBI exposure. In conclusion, VND3207 promoted intestinal repair following radiation injury by reducing reactive oxygen species-induced DNA damage and modulating appropriate levels of activated p53 in intestinal epithelial cells.
Asunto(s)
Benzaldehídos/farmacología , Microbioma Gastrointestinal/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores Acoplados a Proteínas G/genética , Proteína p53 Supresora de Tumor/genética , Animales , Antioxidantes/farmacología , Benzaldehídos/química , Linaje de la Célula/efectos de los fármacos , Linaje de la Célula/efectos de la radiación , Microbioma Gastrointestinal/efectos de la radiación , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Intestinos/efectos de los fármacos , Ratones , Estrés Oxidativo/efectos de los fármacos , Exposición a la Radiación/efectos adversos , Traumatismos por Radiación/genética , Traumatismos por Radiación/prevención & control , Tolerancia a Radiación/genética , Transducción de Señal/efectos de la radiación , Células Madre/efectos de los fármacosRESUMEN
Therapeutic drug synergism intervened in cancer treatments has been demonstrated to be more effective than using a single effector. However, it remains inherently challenging, with a limited cell count from tumor samples, to achieve potent personalized drug cocktails. To address the issue above, we herein present a nanodroplet cell processing platform. The platform incorporates an automatic nanodroplet dispenser with cell array ParaStamp chips, which were fabricated by a new wax stamping approach derived from laser direct writing. Such approach enables not only the on-demand de-wetting with hydrophobic wax films on substrates but also the mask-less fabrication of non-planar microstructures (i.e. no photolithography process). The ParaStamp chip was pre-occupied with anti-cancer drugs and their associate mixtures, enabling for the spatially addressable screening of optimal drug combinations simultaneously. Each droplet with a critical volume of 200 nl containing with 100 cells was utilized. Results revealed that the optimal combination reduces approximate 28-folds of conducted doses compared with single drugs. Tumor inhibition with the optimally selected drug combination was further confirmed by using PC-3 tumor-bearing mouse models. Together, the nanodroplet cell processing platform could therefore offer new opportunities to power the personalized cancer medicine at early-stage drug screening and discovery.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Ensayos de Selección de Medicamentos Antitumorales/métodos , Animales , Dimetilpolisiloxanos , Sinergismo Farmacológico , Diseño de Equipo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Rayos Láser , Masculino , Ratones Desnudos , Miniaturización , Células PC-3 , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Further analysis has revealed that the signal reported in Extended Data Fig. 1c of this Letter is attributed to phosphorylethanolamine, not carbamoyl phosphate. A newly developed derivatization method revealed that the level of carbamoyl phosphate in these NSCLC extracts is below the detection threshold of approximately 10 nanomoles. These findings do not alter the overall conclusions of the Letter; see associated Amendment for full details. The Letter has not been corrected online.
RESUMEN
PURPOSE: Renal cell carcinoma (RCC) is known to be highly radioresistant but the mechanisms associated with radioresistance have remained elusive. We found DOC-2/DAB2 interactive protein (DAB2IP) frequently downregulated in RCC, is associated with radioresistance. In this study, we investigated the underlying mechanism regulating radioresistance by DAB2IP and developed appropriate treatment. EXPERIMENTAL DESIGN: Several RCC lines with or without DAB2IP expression were irradiated with ionizing radiation (IR) for determining their radiosensitivities based on colony formation assay. To investigate the underlying regulatory mechanism of DAB2IP, immunoprecipitation-mass spectrometry was performed to identify DAB2IP-interactive proteins. PARP-1 expression and enzymatic activity were determined using qRT-PCR, Western blot analysis, and ELISA. In vivo ubiquitination assay was used to test PARP-1 degradation. Furthermore, in vivo mice xenograft model and patient-derived xenograft (PDX) model were used to determine the effect of combination therapy to sensitizing tumors to IR. RESULTS: We notice that DAB2IP-deficient RCC cells acquire IR-resistance. Mechanistically, DAB2IP can form a complex with PARP-1 and E3 ligases that is responsible for degrading PARP-1. Indeed, elevated PARP-1 levels are associated with the IR resistance in RCC cells. Furthermore, PARP-1 inhibitor can enhance the IR response of either RCC xenograft model or PDX model. CONCLUSIONS: In this study, we unveil that loss of DAB2IP resulted in elevated PARP-1 protein is associated with IR-resistance in RCC. These results provide a new targeting strategy to improve the efficacy of radiotherapy of RCC.