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1.
Cell Death Dis ; 15(5): 373, 2024 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-38811535

RESUMEN

The targeted elimination of radio- or chemotherapy-induced senescent cells by so-called senolytic substances represents a promising approach to reduce tumor relapse as well as therapeutic side effects such as fibrosis. We screened an in-house library of 178 substances derived from marine sponges, endophytic fungi, and higher plants, and determined their senolytic activities towards DNA damage-induced senescent HCT116 colon carcinoma cells. The Pan-PI3K-inhibitor wortmannin and its clinical derivative, PX-866, were identified to act as senolytics. PX-866 potently induced apoptotic cell death in senescent HCT116, MCF-7 mammary carcinoma, and A549 lung carcinoma cells, independently of whether senescence was induced by ionizing radiation or by chemotherapeutics, but not in proliferating cells. Other Pan-PI3K inhibitors, such as the FDA-approved drug BAY80-6946 (Copanlisib, Aliqopa®), also efficiently and specifically eliminated senescent cells. Interestingly, only the simultaneous inhibition of both PI3K class I alpha (with BYL-719 (Alpelisib, Piqray®)) and delta (with CAL-101 (Idelalisib, Zydelig®)) isoforms was sufficient to induce senolysis, whereas single application of these inhibitors had no effect. On the molecular level, inhibition of PI3Ks resulted in an increased proteasomal degradation of the CDK inhibitor p21WAF1/CIP1 in all tumor cell lines analyzed. This led to a timely induction of apoptosis in senescent tumor cells. Taken together, the senolytic properties of PI3K-inhibitors reveal a novel dimension of these promising compounds, which holds particular potential when employed alongside DNA damaging agents in combination tumor therapies.


Asunto(s)
Senescencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Humanos , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células HCT116 , Complejo de la Endopetidasa Proteasomal/metabolismo , Apoptosis/efectos de los fármacos , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Células MCF-7 , Proteolisis/efectos de los fármacos , Células A549 , Wortmanina/farmacología , Senoterapéuticos/farmacología , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/genética , Daño del ADN/efectos de los fármacos , Pirimidinas , Quinazolinas
2.
Cells ; 12(1)2022 12 23.
Artículo en Inglés | MEDLINE | ID: mdl-36611854

RESUMEN

Chronic liver diseases including hepatocellular carcinoma (HCC) create a state of chronic inflammation that affects the brain via the liver-brain axis leading to an alteration of neurotransmission and cognition. However, little is known about the effects of HCC on the hippocampus, the key brain region for learning and memory. Moreover, radiotherapy used to treat HCC has severe side effects that impair patients' life quality. Thus, designing optimal strategies, such as chronotherapy, to enhance the efficacy and reduce the side effects of HCC treatment is critically important. We addressed the effects of HCC and the timed administration of radiotherapy in mice on the expression of pro-inflammatory cytokines, clock genes, markers for glial activation, oxidative stress, neuronal activity and proliferation in the hippocampal neurogenic niche. Our data showed that HCC induced the upregulation of genes encoding for pro-inflammatory cytokines, altered clock gene expressions and reduced proliferation in the hippocampus. Radiotherapy, in particular when applied during the light/inactive phase enhanced all these effects in addition to glial activation, increased oxidative stress, decreased neuronal activity and increased levels of phospho(p)-ERK. Our results suggested an interaction of the circadian molecular clockwork and the brain's innate immune system as key players in liver-brain crosstalk in HCC and that radiotherapy when applied during the light/inactive phase induced the most profound alterations in the hippocampus.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Ratones , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/radioterapia , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/tratamiento farmacológico , Citocinas/metabolismo , Hipocampo/metabolismo
3.
Cancer Med ; 10(21): 7712-7725, 2021 11.
Artículo en Inglés | MEDLINE | ID: mdl-34545699

RESUMEN

This study investigates whether a chronotherapeutic treatment of hepatocellular carcinoma (HCC) may improve treatment efficacy and mitigate side effects on non-tumoral liver (NTL). HCC was induced in Per2::luc mice which were irradiated at four time points of the day. Proliferation and DNA-double strand breaks were analyzed in irradiated and nonirradiated animals by detection of Ki67 and γ-H2AX. Prior to whole animal experiments, organotypic slice cultures were investigated to determine the dosage to be used in whole animal experiments. Irradiation was most effective at the proliferation peaks in HCC at ZT02 (early inactivity phase) and ZT20 (late activity phase). Irradiation effects on NTL were minimal at ZT20. As compared with NTL, nonirradiated HCC revealed disruption in daily variation and downregulation of all investigated clock genes except Per1. Irradiation affected rhythmic clock gene expression in NTL and HCC at all ZTs except at ZT20 (late activity phase). Irradiation at ZT20 had no effect on total leukocyte numbers. Our results indicate ZT20 as the optimal time point for irradiation of HCC in mice at which the ratio between efficacy of tumor treatment and toxic side effects was maximal. Translational studies are now needed to evaluate whether the late activity phase is the optimal time point for irradiation of HCC in man.


Asunto(s)
Carcinoma Hepatocelular/radioterapia , Cronoterapia , Neoplasias Hepáticas/radioterapia , Animales , Recuento de Células Sanguíneas , Proteínas CLOCK/genética , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proliferación Celular , Daño del ADN , Regulación hacia Abajo , Expresión Génica , Histonas/análisis , Antígeno Ki-67/análisis , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Cultivo de Órganos , Factores de Tiempo
4.
J Pineal Res ; 70(3): e12724, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33615553

RESUMEN

Cancer-related fatigue (CRF) and stress are common symptoms in cancer patients and represent early side effects of cancer treatment which affect the life quality of the patients. CRF may partly depend on disruption of the circadian rhythm. Locomotor activity and corticosterone rhythms are two important circadian outputs which can be used to analyze possible effects on the circadian function during cancer development and treatment. The present study analyzes the relationship between locomotor activity rhythm, corticosterone levels, hepatocellular carcinoma (HCC) development, and radiotherapy treatment in a mouse model. HCC was induced in mice by single injection of diethylnitrosamine (DEN) and chronic treatment of phenobarbital in drinking water. Another group received chronic phenobarbital treatment only. Tumor bearing animals were divided randomly into four groups irradiated at four different Zeitgeber time points. Spontaneous locomotor activity was recorded continuously; serum corticosterone levels and p-ERK immunoreaction in the suprachiasmatic nucleus (SCN) were investigated. Phenobarbital treated mice showed damped corticosterone levels and a less stable 24 hours activity rhythm as well as an increase in activity during the light phase, reminiscent of sleep disruption. The tumor mice showed an increase in corticosterone level during the inactive phase and decreased activity during the dark phase, reminiscent of CRF. After irradiation, corticosterone levels were further increased and locomotor activity rhythms were disrupted. Lowest corticosterone levels were observed after irradiation during the early light phase; thus, this time might be the best to apply radiotherapy in order to minimize side effects.


Asunto(s)
Ciclos de Actividad , Conducta Animal , Carcinoma Hepatocelular/radioterapia , Ritmo Circadiano , Corticosterona/sangre , Neoplasias Hepáticas Experimentales/radioterapia , Locomoción , Núcleo Supraquiasmático/fisiopatología , Animales , Biomarcadores/sangre , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/fisiopatología , Cronoterapia , Dietilnitrosamina , Progresión de la Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neoplasias Hepáticas Experimentales/sangre , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Circadianas Period/genética , Fenobarbital , Fosforilación , Núcleo Supraquiasmático/metabolismo , Factores de Tiempo
5.
Cells ; 9(9)2020 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-32846968

RESUMEN

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD+-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G2 and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,ß-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus.


Asunto(s)
Centrosoma/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/metabolismo , Mitosis/genética , Sirtuinas/metabolismo , Humanos
6.
Cell Cycle ; 19(4): 464-478, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31959066

RESUMEN

Microtubule-targeting agents (MTAs) are the most effective chemotherapeutics used in cancer therapy to date, but their clinical use is often hampered by the acquisition of resistance. Thereby, elucidation of the molecular signaling pathways activated by novel FDA-approved MTAs such as eribulin is important for future therapeutic applications. In contrast to several reports, we show here that regardless of the presence of caspase-3, clinically relevant concentrations of eribulin and the classical MTA paclitaxel predominantly induce caspase-independent cell death in MCF-7 breast carcinoma cells. On the molecular level, several key proteins involved in apoptosis such as p53, Plk1, caspase-2, and Bim as well as the two MAPKs ERK and JNK were activated by both compounds to a similar extent. However, none of them proved to be important for eribulin- and paclitaxel-induced cytotoxicity, as their siRNA-mediated knockdown or inactivation by small molecule inhibitors did not alter cell death rates. In contrast, knockdown of the anti-apoptotic Bcl-2 protein, which becomes heavily phosphorylated at Ser70 during MTA treatment, resulted surprisingly in a reduction of MTA-mediated cell death. This phenomenon can be most likely explained by our observation that the absence of Bcl-2 slowed down cell cycle progression resulting in fewer cells entering mitosis, thereby delaying the mitotic capability of these MTAs to induce cell death. Taken together, although eribulin and paclitaxel disturb the mitotic spindle differently, they exhibit no functional differences in downstream molecular cell death signaling in MCF-7 breast cancer cells.


Asunto(s)
Caspasa 3/metabolismo , Furanos/farmacología , Cetonas/farmacología , Microtúbulos/metabolismo , Paclitaxel/farmacología , Transducción de Señal , Antracenos/farmacología , Butadienos/farmacología , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Microtúbulos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Nitrilos/farmacología , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Transducción de Señal/efectos de los fármacos
7.
Cell Death Differ ; 27(4): 1274-1285, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31511650

RESUMEN

In recent years it has become more and more apparent that the regulation of gene expression by RNA-binding proteins (RBPs) is of utmost importance for most cellular signaling pathways. RBPs control several aspects of RNA biogenesis including splicing, localization, stability, and translation efficiency. One of these RBPs is RBM47 that recently has been suggested to function as a tumor suppressor as it was shown to suppress breast and colon cancer progression. Here we demonstrate that RBM47 is an important regulator of basal and DNA damage-induced p53 and p21WAF1/CIP1 protein expression. Knockdown of RBM47 by siRNAs results in a strong reduction in p53 mRNA and protein levels due to an impaired p53 promoter activity. Accordingly, overexpression of Flag-RBM47 enhances p53 promoter activity demonstrating that RBM47 regulates p53 at the transcriptional level. By controlling p53, knockdown of RBM47 concomitantly decreases also p21 expression at the transcriptional level, driving irradiated carcinoma cell lines from different entities into cell death rather than into senescence. Thus, RBM47 represents a novel molecular switch of cell fate decisions that functions as a regulator of the p53/p21-signaling axis.


Asunto(s)
Linaje de la Célula/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Regiones Promotoras Genéticas , Proteína p53 Supresora de Tumor/metabolismo
8.
Endosc Int Open ; 7(9): E1175-E1183, 2019 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-31475237

RESUMEN

Background and study aims Our academic lab has developed a novel, low-cost, disposable endoscope for assessment of the esophagus and stomach without need for large equipment or complex electronics. Usability and intuitiveness of the platform are unknown. Methods The novel endoscope (NE) consists of a high-definition camera, LED module, and three bellows. Compressed air actuates the bellows, producing camera/LED articulation. Insufflation and lens cleaning ports are present. Video can be displayed on any monitor. Total material costs less than $ 35 US. Five novices, five fellows, and five attendings performed five trials using a conventional endoscope and the NE on an upper tract phantom with six gastric landmarks marked. Outcomes included successful identification and time to landmarks; and intuitiveness (NASA task load index; user comments). Results All landmarks were successfully identified with both endoscopes for all trials (n = 900). Attendings and fellows were quicker with the conventional endoscope when compared to the NE (24.48 v 37.13s; P  < 0.01). There was no significant time difference between platforms for novices ( P  = 0.16). All users found the NE intuitive with low mental and physical demand. Novices reported lower temporal demand and effort when using the NE. Conclusions The NE was easy to maneuver, intuitive, and successful at visualizing gastric landmarks. All users were pleased with the NE drive mechanism and were successful at visualizing the gastric landmarks in a clinically acceptable time. The novel platform has the potential to facilitate rapid, low-cost, diagnostic assessment of the esophagus and stomach in non-traditional settings - facilitating patient management decisions, minimizing encumbrance, and avoiding cross-contamination.

9.
Haematologica ; 104(1): 35-46, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30093397

RESUMEN

The homeobox gene HLXB9 encodes for the transcription factor HB9, which is essential for pancreatic as well as motor neuronal development. Beside its physiological expression pattern, aberrant HB9 expression has been observed in several neoplasias. Especially in infant translocation t(7;12) acute myeloid leukemia, aberrant HB9 expression is the only known molecular hallmark and is assumed to be a key factor in leukemic transformation. However, so far, only poor functional data exist addressing the oncogenic potential of HB9 or its influence on hematopoiesis. We investigated the influence of HB9 on cell proliferation and cell cycle in vitro, as well as on hematopoietic stem cell differentiation in vivo using murine and human model systems. In vitro, HB9 expression led to premature senescence in human HT1080 and murine NIH3T3 cells, providing for the first time evidence for an oncogenic potential of HB9. Onset of senescence was characterized by induction of the p53-p21 tumor suppressor network, resulting in growth arrest, accompanied by morphological transformation and expression of senescence-associated ß-galactosidase. In vivo, HB9-transduced primary murine hematopoietic stem and progenitor cells underwent a profound differentiation arrest and accumulated at the megakaryocyte/erythrocyte progenitor stage. In line, gene expression analyses revealed de novo expression of erythropoiesis-related genes in human CD34+hematopoietic stem and progenitor cells upon HB9 expression. In summary, the novel findings of HB9-dependent premature senescence and myeloid-biased perturbed hematopoietic differentiation, for the first time shed light on the oncogenic properties of HB9 in translocation t(7;12) acute myeloid leukemia.


Asunto(s)
Ciclo Celular , Diferenciación Celular , Senescencia Celular , Regulación Leucémica de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Proteínas de Homeodominio/biosíntesis , Leucemia Mieloide Aguda/metabolismo , Proteínas de Neoplasias/biosíntesis , Factores de Transcripción/biosíntesis , Animales , Eritropoyesis/genética , Células Madre Hematopoyéticas/patología , Proteínas de Homeodominio/genética , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Ratones , Células 3T3 NIH , Proteínas de Neoplasias/genética , Factores de Transcripción/genética , Translocación Genética
10.
J Biol Chem ; 292(20): 8331-8341, 2017 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-28348086

RESUMEN

The cyclin-dependent kinase inhibitor p21 is an important player in stress pathways exhibiting both tumor-suppressive and oncogenic functions. Thus, expression of p21 has to be tightly controlled, which is achieved by numerous mechanisms at the transcriptional, translational, and posttranslational level. Performing immunoprecipitation of bromouridine-labeled p21 mRNAs that had been incubated before with cytoplasmic extracts of untreated HCT116 colon carcinoma cells, we identified the DEAD-box RNA helicase DDX41 as a novel regulator of p21 expression. DDX41 specifically precipitates with the 3'UTR, but not with the 5'UTR, of p21 mRNA. Knockdown of DDX41 increases basal and γ irradiation-induced p21 protein levels without affecting p21 mRNA expression. Conversely, overexpression of DDX41 strongly inhibits expression of a FLAG-p21 and a luciferase construct, but only in the presence of the p21 3'UTR. Together, these data suggest that this helicase regulates p21 expression at the translational level independent of the transcriptional activity of p53. However, knockdown of DDX41 completely fails to increase p21 protein levels in p53-deficient HCT116 cells. Moreover, posttranslational up-regulation of p21 achieved in both p53+/+ and p53-/- HCT116 cells in response to pharmaceutical inhibition of the proteasome (by MG-132) or p90 ribosomal S6 kinases (by BI-D1870) is further increased by knockdown of DDX41 only in p53-proficient but not in p53-deficient cells. Although our data demonstrate that DDX41 suppresses p21 translation without disturbing the function of p53 to directly induce p21 mRNA expression, this process indirectly requires p53, perhaps in the form of another p53 target gene or as a still undefined posttranscriptional function of p53.


Asunto(s)
Regiones no Traducidas 3'/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , ARN Helicasas DEAD-box/metabolismo , Biosíntesis de Proteínas/fisiología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , ARN Helicasas DEAD-box/genética , Técnicas de Silenciamiento del Gen , Humanos , Biosíntesis de Proteínas/efectos de los fármacos , Pteridinas/farmacología , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
11.
Support Care Cancer ; 24(10): 4241-8, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27165055

RESUMEN

BACKGROUND: The mechanisms of skin aging have not been completely elucidated. Anecdotal data suggests that EGFR inhibition accelerates aging-like skin changes. OBJECTIVE: The objective of the study was to evaluate the clinical characteristics and investigate the cellular and molecular mechanisms underlying skin changes associated with the use of EFGRIs. PATIENTS AND METHODS: Patients during prolonged treatment with EGFRIs (>3 months) were analyzed for aging-like skin changes. Baseline EGFR expression was compared in young (<25 years old) vs. old (> 65 years old) skin. In addition, the regulation of extracellular matrix, senescence-associated genes, and cell cycle status was measured in primary human keratinocytes treated with erlotinib in vitro. RESULTS: There were progressive signs of skin aging, including xerosis cutis, atrophy, rhytide formation, and/or actinic purpura in 12 patients. Keratinocytes treated with erlotinib in vitro showed a significant down-modulation of hyaluronan synthases (HAS2 and HAS3), whereas senescence-associated genes (p21, p53, IL-6, maspin) were upregulated, along with a G1 cell cycle arrest and stronger SA ß-Gal activity. There was significantly decreased baseline expression in EGFR density in aged skin, when compared to young controls. CONCLUSIONS: EGFR inhibition results in molecular alterations in keratinocytes that may contribute to the observed skin aging of patients treated with respective targeted agents.


Asunto(s)
Receptores ErbB/genética , Clorhidrato de Erlotinib/uso terapéutico , Envejecimiento de la Piel/genética , Enfermedades de la Piel/genética , Anciano , Anciano de 80 o más Años , Envejecimiento , Receptores ErbB/antagonistas & inhibidores , Clorhidrato de Erlotinib/administración & dosificación , Clorhidrato de Erlotinib/farmacología , Femenino , Humanos , Estudios Retrospectivos
12.
Oncotarget ; 7(13): 15915-29, 2016 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-26895377

RESUMEN

MicroRNAs (miRNAs), a class of small non-coding RNAs that usually cause gene silencing by translational repression or degradation of mRNAs, are implicated in DNA damage-induced stress responses. To identify senescence-associated miRNAs, we performed microarray analyses using wild-type and p53-deficient HCT116 colon carcinoma cells that following gamma-irradiation (γIR) are driven into senescence and apoptosis, respectively. Several miRNAs including miR-30e were found upregulated in a p53-dependent manner specifically in senescent cells, but not in apoptotic cells. Overexpression of miR-30e in HCT116 cells not only inhibited γIR-, etoposide- or miR-34a-induced caspase-3-like DEVDase activities and cell death, but greatly accelerated and augmented their senescent phenotype. Consistently, procaspase-3 protein, but not mRNA decreased in the presence of miR-30e, whereas expression of the cyclin-dependent kinase inhibitor p21 increased both at the mRNA and protein level. Performing luciferase reporter gene assays, we identified the 3'-UTR of the caspase-3 mRNA as a direct miR-30e target. In contrast, although miR-30e was unable to bind to the p21 mRNA, it increased expression of a luciferase construct containing the p21 promoter, suggesting that the miR-30e-mediated upregulation of p21 occurs indirectly at the transcriptional level. Interestingly, despite suppressing procaspase-3 expression, miR-30e was unable to protect RKO colon carcinoma cells from DNA damage-induced death or to induce senescence, as miR-30e completely fails to upregulate p21 in these cells. These data suggest that miR-30e functions in a cell type-dependent manner as an important molecular switch for DNA damage-induced stress responses and may thus represent a target of therapeutic value.


Asunto(s)
Caspasa 3/biosíntesis , Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Daño del ADN/fisiología , MicroARNs/metabolismo , Regulación de la Expresión Génica , Células HCT116 , Humanos
13.
PLoS One ; 8(4): e61438, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23593480

RESUMEN

Proteasome inhibitors (PIs) potently induce apoptosis in a variety of tumor cells, but the underlying mechanisms are not fully elucidated. Comparing PI-induced apoptosis susceptibilities of various mouse embryonic fibroblast (MEF) lines differing in their c-jun N-terminal kinase (JNK) 1 and 2 status, we show that several hallmarks of apoptosis were most rapidly detectable in JNK2-/- cells, whereas they appeared only delayed and severely reduced in their intensities in cells expressing JNK2. Consistent with our finding that PI-induced apoptosis requires de novo protein synthesis, the proteasomal inhibitor MG-132 induced expression of the BH3-only protein Noxa at the transcriptional level in a JNK1-dependent, but JNK2-opposing manner. As the knockdown of Noxa blocked only the rapid PI-induced apoptosis of JNK2-/- cells, but not the delayed death occurring in JNK1-/- and JNK1+/+ cells, our data uncover a novel PI-induced apoptosis pathway that is regulated by the JNK1/2-dependent expression of Noxa. Furthermore, several transcription factors known to modulate Noxa expression including ATF3, ATF4, c-Jun, c-Myc, HIF1α, and p53 were found upregulated following MG-132 exposure. From those, only knockdown of c-Myc rescued JNK2-/- cells from PI-induced apoptosis, however, without affecting expression of Noxa. Together, our data not only show that a rapid execution of PI-induced apoptosis requires JNK1 for upregulation of Noxa via an as yet unknown transcription factor, but also that JNK2 controls this event in an oppositional manner.


Asunto(s)
Apoptosis/fisiología , Regulación de la Expresión Génica/fisiología , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Inhibidores de Proteasoma/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Línea Celular , Fluorometría , Técnicas de Inactivación de Genes , Leupeptinas , Ratones , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/genética , Reacción en Cadena de la Polimerasa
14.
J Am Soc Nephrol ; 22(12): 2193-202, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22034640

RESUMEN

The prorenin receptor (PRR) is highly expressed in podocytes, but its role in the maintenance of podocyte function is unknown. Here we generated podocyte-specific PRR-knockout mice and found that these animals died between 2 to 3 wk after birth. Within 14 d, PRR-knockout mice developed nephrotic syndrome, albuminuria with podocyte foot-process fusion, and cytoskeletal changes. Podocyte-specific PRR deletion also led to disturbed processing of multivesicular bodies and enrichment of autophagosomal (LC3) and lysosomal (LAMP2) markers, indicating a functional block in autophagosome-lysosome fusion and an overload of the proteasomal protein-degradation machinery. In vitro, PRR knockdown and pharmacologic blockade of vacuolar H(+)-ATPases, which associate with the PRR, increased vesicular pH, led to accumulation of LC3-positive and LAMP2-positive vesicles and altered the cytoskeleton. Taken together, these results suggest that the PRR is essential for podocyte function and survival by maintaining autophagy and protein-turnover machinery. Furthermore, PRR contributes to the control of lysosomal pH, which is important for podocyte survival and cytoskeletal integrity.


Asunto(s)
Autofagia/fisiología , Podocitos/fisiología , Receptores de Superficie Celular/fisiología , Animales , Supervivencia Celular , Femenino , Ratones , Receptor de Prorenina
15.
Cell Cycle ; 9(17): 3575-83, 2010 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-20818156

RESUMEN

Although initially described as a regulator of cell cycle progression, the cyclin-dependent kinase inhibitor p21 is now known to also modulate various other biological processes including transcription, differentiation and apoptosis. These versatile activities of p21 are mainly mediated via direct binding to various transcription factors, pro-apoptotic proteins and kinases that are usually inhibited by this interaction. Here we provide in vitro evidence that p21 not only inhibits, but also activates certain kinases in a remarkable substrate-dependent manner. Whereas phosphorylation of the tumor suppressor p53 by several isoforms of the cJun N-terminal kinases (JNKs) was greatly attenuated in the presence of p21, phosphorylation of cJun remained either unaffected or was even enhanced. Furthermore, p21 strongly increased phosphorylation of cFos and MBP by ERK1 and ERK2, while p53 phosphorylation was increased and inhibited, respectively. Also p38α and glycogen synthase kinase-3 beta (GSK-3ß) were found differentially regulated by p21 in a substrate-dependent manner, while casein kinase-1 epsilon (CK1ε) was not affected. Together with our finding that the stress-induced p53 phosphorylation pattern differs greatly between p21-proficient and -deficient HCT116 colon carcinoma cells, our results suggest that p21 is able to influence kinase activities both in a negative and positive manner.


Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Básica de Mielina , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Transcripción/metabolismo
16.
Cell Cycle ; 6(4): 407-13, 2007 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-17312393

RESUMEN

Until recently, the p53 transcriptional target p21, a member of the Cip/Kip family, was almost solely viewed as a nuclear protein with a principal function of inhibiting cyclin-dependent kinase (CDK) activity and hence, cell cycle progression. However, emerging evidence now suggests additional functions for p21 in diverse cellular processes including a role as a modulator of apoptosis. Several mechanisms are suggested by which p21 interferes with the apoptotic machinery that encompass either CDK-independent events such as transcriptional regulation and direct binding to pro-apoptotic gene products in the cytoplasm, or that were based on inhibition of CDKs that in several studies were required downstream of caspases for the generation of characteristic apoptotic alterations. Very recently, we have shown that p21 protects cells from irradiation-induced apoptosis by suppression of CDK activity that appears to be required downstream of the mitochondria for an efficient activation of the caspase cascade. Together with other reports, our results not only demonstrate the close connection of events leading to either cell cycle arrest or apoptosis, but also indicate that the CDK inhibitor p21 battles apoptosis at multiple frontiers.


Asunto(s)
Apoptosis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Transducción de Señal , Caspasas/metabolismo , Ciclo Celular/fisiología , Línea Celular Tumoral , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Daño del ADN , Regulación de la Expresión Génica , Humanos , Modelos Biológicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
17.
Mol Biol Cell ; 18(1): 84-93, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17079734

RESUMEN

Caspase-9 plays an important role in apoptosis induced by genotoxic stress. Irradiation and anticancer drugs trigger mitochondrial outer membrane permeabilization, resulting in cytochrome c release and caspase-9 activation. Two highly contentious issues, however, remain: It is unclear whether the loss of the mitochondrial membrane potential DeltaPsi(M) contributes to cytochrome c release and whether caspases are involved. Moreover, an unresolved question is whether caspase-2 functions as an initiator in genotoxic stress-induced apoptosis. In the present study, we have identified a mutant Jurkat T-cell line that is deficient in caspase-9 and resistant to apoptosis. Anticancer drugs, however, could activate proapoptotic Bcl-2 proteins and cytochrome c release, similarly as in caspase-9-proficient cells. Interestingly, despite these alterations, the cells retained DeltaPsi(M). Furthermore, processing and enzyme activity of caspase-2 were not observed in the absence of caspase-9. Reconstitution of caspase-9 expression restored not only apoptosis but also the loss of DeltaPsi(M) and caspase-2 activity. Thus, we provide genetic evidence that caspase-9 is indispensable for drug-induced apoptosis in cancer cells. Moreover, loss of DeltaPsi(M) can be functionally separated from cytochrome c release. Caspase-9 is not only required for DeltaPsi(M) loss but also for caspase-2 activation, suggesting that these two events are downstream of the apoptosome.


Asunto(s)
Caspasa 2/metabolismo , Caspasa 9/deficiencia , Caspasa 9/metabolismo , Potencial de la Membrana Mitocondrial/fisiología , Membranas Mitocondriales/metabolismo , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cromatina/ultraestructura , Citocromos c/metabolismo , Resistencia a Antineoplásicos , Activación Enzimática/efectos de los fármacos , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Células Jurkat , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/ultraestructura , Mutágenos/farmacología , Estaurosporina/farmacología , Transfección , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo
18.
Cancer Res ; 66(23): 11254-62, 2006 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-17145870

RESUMEN

The role of the cyclin-dependent kinase (CDK) inhibitor p21 as a mediator of p53-induced growth arrest is well established. In addition, recent data provide strong evidence for new emerging functions of p21, including a role as a modulator of apoptosis. The mechanisms, however, by which p21 interferes with the death machinery, especially following ionizing radiation (IR), are largely unknown. Here, we report that IR induced caspase-9 and caspase-3 activation and subsequent apoptosis only in p21-deficient colon carcinoma cells, whereas similar treated wild-type cells were permanently arrested in the G(2)-M phase, correlating with the induction of cellular senescence. Interestingly, activation of the mitochondrial pathway, including caspase-2 processing, depolarization of the outer mitochondrial membrane, and cytochrome c release, was achieved by IR in both cell lines, indicating that p21 inhibits an event downstream of mitochondria but preceding caspase-9 activation. IR-induced p21 protein expression was restricted to the nucleus, and no evidence for a mitochondrial or cytoplasmic association was found. In addition, p21 did neither interact with caspase-3 or caspase-9, suggesting that these events are not required for the observed protection. Consistent with this assumption, we found that CDK inhibitors potently abrogated IR-induced caspase processing and activation without affecting mitochondrial events. In addition, in vitro caspase activation assays yielded higher caspase-3 activities in extracts of irradiated p21-deficient cells compared with extracts of similar treated wild-type cells. Thus, our results strongly indicate that p21 protects cells from IR-induced apoptosis by suppression of CDK activity that seems to be required for activation of the caspase cascade downstream of the mitochondria.


Asunto(s)
Apoptosis/efectos de la radiación , Caspasa 9/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Mitocondrias/efectos de la radiación , Western Blotting , Caspasa 3/metabolismo , Inhibidores de Caspasas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/deficiencia , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Genotipo , Células HCT116 , Humanos , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/fisiología , Mutación/genética , Oligopéptidos/farmacología , Unión Proteica/efectos de la radiación , Inhibidores de Proteínas Quinasas/farmacología , Purinas/farmacología , Radiación Ionizante , Roscovitina , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
20.
Cell Cycle ; 5(8): 841-5, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16628002

RESUMEN

The proteasome is a multicatalytic enzyme complex that is responsible for degradation of the vast majority of intracellular proteins. Thus, it is involved in diverse cellular processes such as proliferation, differentiation and apoptosis. Especially its latter function yielded in the development of specific proteasomal inhibitors which have recently entered clinical trials due to their tremendous apoptosis-inducing capability. However, several recent studies including ours provided substantial evidence that a combined treatment of tumors with apoptosis-inducing agents and proteasomal inhibitors might even cause adverse effects leading to a prolonged survival of tumor cells. Based on our model of a biphasic role for the proteasome in apoptosis, we believe that a successful combat of tumors that relies on a combinational therapy with proteasomal inhibitors requires careful evaluation of several critical aspects in order to avoid a friend becoming a foe.


Asunto(s)
Resistencia a Antineoplásicos , Neoplasias/patología , Complejo de la Endopetidasa Proteasomal/fisiología , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis , Diferenciación Celular , Terapia Combinada , Humanos , Neoplasias/tratamiento farmacológico , Complejo de la Endopetidasa Proteasomal/metabolismo
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