RESUMEN
The tumor-suppressor p53 is a transcription factor that prevents cancer development and is involved in regulation of various physiological processes. This is mediated both by induction of cell cycle arrest and apoptosis and by controlling the expression of a plethora of target genes, including secreted proteins. It has been demonstrated that p53 may exert its effect in non-cell-autonomous manner by modulating the expression of genes that encode for secreted factors. In this study, we utilized our microarray data to identify and characterize novel p53 target genes expressed in human liver cells and associated with steroid hormones processing and transfer. We identified the steroid hormones binding factors, sex hormone-binding globulin (SHBG), corticosteroid-binding globulin (CBG) and cytochrome P450 family 21 subfamily A polypeptide 2, as novel p53 target genes. Their expression and secretion was increased following p53 activation in various hepatic cells. We observed that p53 wild-type mice exhibited higher levels of CBG compared with their p53 null counterparts. We demonstrated that the induction of the steroid hormones binding factors can be mediated by binding to specific p53 responsive elements within their promoters. In addition, utilizing conditioned medium experiments we have shown that p53-dependent induction of SHBG secretion from liver cells enhances apoptosis of breast cancer cells. Moreover, depletion of SHBG abolished the induction of breast cancer cells death. The newly identified p53 target genes suggest a novel non-cell-autonomous tumor-suppressive regulation mediated by p53 that is central for maintaining organism homeostasis.
Asunto(s)
Hígado/metabolismo , Activación Transcripcional , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis , Células Hep G2 , Humanos , Células MCF-7 , Ratones Endogámicos C57BL , Ratones Noqueados , Globulina de Unión a Hormona Sexual/genética , Globulina de Unión a Hormona Sexual/metabolismo , Esteroide 21-Hidroxilasa/genética , Esteroide 21-Hidroxilasa/metabolismo , Transcortina/genética , Transcortina/metabolismo , Transcripción GenéticaRESUMEN
p53 loss of heterozygosity (p53LOH) is frequently observed in Li-Fraumeni syndrome (LFS) patients who carry a mutant (Mut) p53 germ-line mutation. Here, we focused on elucidating the link between p53LOH and tumor development in stem cells (SCs). Although adult mesenchymal stem cells (MSCs) robustly underwent p53LOH, p53LOH in induced embryonic pluripotent stem cells (iPSCs) was significantly attenuated. Only SCs that underwent p53LOH induced malignant tumors in mice. These results may explain why LFS patients develop normally, yet acquire tumors in adulthood. Surprisingly, an analysis of single-cell sub-clones of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that p53LOH is a bi-directional process, which may result in either the loss of wild-type (WT) or Mut p53 allele. Interestingly, most BM progenitors underwent Mutp53LOH. Our results suggest that the bi-directional p53LOH process may function as a cell-fate checkpoint. The loss of Mutp53 may be regarded as a DNA repair event leading to genome stability. Indeed, gene expression analysis of the p53LOH process revealed upregulation of a specific chromatin remodeler and a burst of DNA repair genes. However, in the case of loss of WTp53, cells are endowed with uncontrolled growth that promotes cancer.
Asunto(s)
Alelos , Pérdida de Heterocigocidad , Células Madre/metabolismo , Proteína p53 Supresora de Tumor/genética , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Proper regulation of white and brown adipogenic differentiation is important for maintaining an organism's metabolic profile in a homeostatic state. The recent observations showing that the p53 tumor suppressor plays a role in metabolism raise the question of whether it is involved in the regulation of white and brown adipocyte differentiation. By using several in vitro models, representing various stages of white adipocyte differentiation, we found that p53 exerts a suppressive effect on white adipocyte differentiation in both mouse and human cells. Moreover, our in vivo analysis indicated that p53 is implicated in protection against diet-induced obesity. In striking contrast, our data shows that p53 exerts a positive regulatory effect on brown adipocyte differentiation. Abrogation of p53 function in skeletal muscle committed cells reduced their capacity to differentiate into brown adipocytes and histological analysis of brown adipose tissue revealed an impaired morphology in both embryonic and adult p53-null mice. Thus, depending on the specific adipogenic differentiation program, p53 may exert a positive or a negative effect. This cell type dependent regulation reflects an additional modality of p53 in maintaining a homeostatic state, not only in the cell, but also in the organism at large.
Asunto(s)
Adipocitos Marrones/metabolismo , Adipogénesis , Tejido Adiposo Pardo/metabolismo , Obesidad/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Células 3T3 , Adipocitos Marrones/citología , Tejido Adiposo Pardo/citología , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Dieta , Metabolismo Energético , Humanos , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Interferencia de ARN , ARN Interferente Pequeño , Factores de Transcripción/metabolismo , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The process of somatic cell reprogramming is gaining increasing interest as reprogrammed cells are considered to hold a great therapeutic potential. However, with current technologies this process is relatively inefficient. Recent studies reported that inhibition of the p53 tumor suppressor profoundly facilitates reprogramming and attributed this effect to the ability of p53 to restrict proliferation and induce apoptosis. Given that mesenchymal-to-epithelial transition (MET) was recently shown to be necessary for reprogramming of fibroblasts, we investigated whether p53 counteracts reprogramming by affecting MET. We found that p53 restricts MET during the early phases of reprogramming and that this effect is primarily mediated by the ability of p53 to inhibit Klf4-dependent activation of epithelial genes. Moreover, transcriptome analysis revealed a large transcriptional signature enriched with epithelial genes, which is markedly induced by Klf4 exclusively in p53(-/-) cells. We also found that the expression of the epithelial marker E-Cadherin negatively correlates with p53 activity in a variety of mesenchymal cells even before the expression of reprogramming factors. Finally, we demonstrate that the inhibitory effect of p53 on MET is mediated by p21. We conclude that inhibition of the p53-p21 axis predisposes mesenchymal cells to the acquisition of epithelial characteristics and renders them more prone to reprogramming. Our study uncovers a novel mechanism by which p53 restrains reprogramming and highlights the role of p53 in regulating cell plasticity.
Asunto(s)
Transición Epitelial-Mesenquimal , Proteína p53 Supresora de Tumor/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Células Cultivadas , Reprogramación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína p53 Supresora de Tumor/antagonistas & inhibidores , Proteína p53 Supresora de Tumor/genéticaRESUMEN
A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53(R175H) mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53(R175H), was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53(R175H) mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53(R175H) mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.
Asunto(s)
Transición Epitelial-Mesenquimal , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 Relacionada con Twist/metabolismo , Sustitución de Aminoácidos , Línea Celular Transformada , Línea Celular Tumoral , Epigénesis Genética , Histonas/metabolismo , Humanos , Masculino , Mutación , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Regiones Promotoras Genéticas , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/fisiología , Proteína 1 Relacionada con Twist/genética , Regulación hacia ArribaRESUMEN
The p53 tumor suppressor serves as a crucial barrier against cancer development. In tumor cells and their progenitors, p53 suppresses cancer in a cell-autonomous manner. However, p53 also possesses non-cell-autonomous activities. For example, p53 of stromal fibroblasts can modulate the spectrum of proteins secreted by these cells, rendering their microenvironment less supportive of the survival and spread of adjacent tumor cells. We now report that epithelial tumor cells can suppress p53 induction in neighboring fibroblasts, an effect reproducible by tumor cell-conditioned medium. The ability to suppress fibroblast p53 activation is acquired by epithelial cells in the course of neoplastic transformation. Specifically, stable transduction of immortalized epithelial cells by mutant H-Ras and p53-specific short inhibitory RNA endows them with the ability to quench fibroblast p53 induction. Importantly, human cancer-associated fibroblasts are more susceptible to this suppression than normal fibroblasts. These findings underscore a mechanism whereby epithelial cancer cells may overcome the non-cell-autonomous tumor suppressor function of p53 in stromal fibroblasts.
Asunto(s)
Fibroblastos/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular , Transformación Celular Neoplásica , Medios de Cultivo Condicionados/farmacología , Células Epiteliales/metabolismo , Genes Supresores de Tumor , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
The tumor suppressor p53 is a key modulator of the cellular stress response, inducing cell-cycle arrest, apoptosis, senescence and cell differentiation. To evaluate further the molecular mechanism underlying p53 function, the transcriptional profiles of proliferating and senescent WI-38 cells, both wild-type p53 expressers and counterparts with an inactivated p53, were compared by DNA microarray analysis. In particular, the amyloid-beta precursor-like protein 1 (APLP1) is induced in senescent cells in a p53-dependent manner. APLP1 was confirmed to be a novel transcriptional target of p53 by in vivo and in vitro characterization of a p53 responsive element found in the first intron of the APLP1 gene locus. APLP1 knockdown experiments demonstrate that APLP1 is required for the proliferation of fibroblastic and epithelial cells. Moreover, depletion of APLP1 expression diminishes stress-induced apoptosis of neural cells, whereas ectopic APLP1 expression augments apoptosis. Based on these data, a mechanism is proposed whereby p53-dependent induction of APLP1 is involved in neural cell death, and which may exacerbate neuronal cell loss in some acute or chronic neurodegenerative disorders.
Asunto(s)
Precursor de Proteína beta-Amiloide/genética , Perfilación de la Expresión Génica , Neuroblastoma/metabolismo , Transcripción Genética , Proteína p53 Supresora de Tumor/metabolismo , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/metabolismo , Muerte Celular , Proliferación Celular , Células Cultivadas , Senescencia Celular/fisiología , Inmunoprecipitación de Cromatina , Fibroblastos/metabolismo , Humanos , Immunoblotting , Luciferasas/metabolismo , Pulmón/embriología , Pulmón/metabolismo , Neuroblastoma/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The tumor suppressor p53 plays a pivotal role in suppressing tumorigenesis by inducing genomic stability, cell cycle arrest or apoptosis. AIF is a mitochondrial protein, which, upon translocation to the nucleus, can participate in apoptosis, primarily in a caspase-independent contexts. We now report that AIF gene expression is subject to positive transcriptional regulation by p53. Interestingly, unlike most known p53 target genes, the AIF gene is regulated by basal levels of p53, and activation of p53 by genotoxic stress does not result in a substantial further increase in AIF expression. The AIF gene harbors a p53 responsive element, which is bound by p53 within cells. p53 drives efficient induction of large-scale DNA fragmentation, a hallmark of AIF activity. Importantly, caspase-independent death is compromised in cells lacking functional p53, in line with the known role of AIF in this process. Thus, in addition to its documented effects on caspase-dependent apoptosis, p53 may also sensitize cells to caspase-independent death through positive regulation of AIF expression. Moreover, in the absence of overt apoptotic signals, the constitutive induction of AIF by p53 may underpin a cytoprotective maintenance role, based on the role of AIF in ensuring proper mitochondrial function.
Asunto(s)
Factor Inductor de la Apoptosis/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Factor Inductor de la Apoptosis/genética , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral , Fragmentación del ADN , ADN de Neoplasias/genética , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Intrones/genética , Intrones/fisiología , Mitocondrias/fisiología , Proteína p53 Supresora de Tumor/genética , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
While it is well accepted that p53 plays a role in apoptosis, less is known as to its involvement in cell differentiation. Here we show that wild-type p53 facilitates IL-6-dependent macrophage differentiation. Treatment of M1/2 cells expressing the temperature-sensitive p53 143 (Val to Ala) mutant, at the wild-type conformation, facilitated the appearance of mature macrophages that exhibited phagocytic activity. Enhancement of differentiation by the p53 143 (Val to Ala) in the wild-type conformation was coupled with the inhibition of apoptosis induction by this protein. In agreement with previous studies, we found that p53 levels were reduced during p53-dependent macrophage differentiation. This occurred when p53 levels before IL-6 stimuli were high. Interestingly, the p53 143 (Val to Ala) protein, at the mutant conformation, enhanced macrophage differentiation, as did the wild-type conformation, whereas the p53 273 (Arg to His) core mutant exerted an inhibitory effect on this pathway. The transcription-deficient p53 molecules, p53 (22-23) and p53 22,23,143, could not induce p53-dependent differentiation. Moreover, the p53 (22-23) protein inhibited the p53-independent differentiation pathway. Interestingly, the p53 (22-23) protein not only blocked IL-6-mediated differentiation, but also induced significant apoptotic cell death, upon IL-6 stimulation. Taken together, our data show that wild-type p53 enhances macrophage differentiation, while various p53 mutant types exert different effects on this differentiation pathway.
Asunto(s)
Diferenciación Celular/fisiología , Macrófagos/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/fisiología , Diferenciación Celular/efectos de los fármacos , Células Clonales , Regulación de la Expresión Génica , Interleucina-6/farmacología , Ratones , Mutación , Células Mieloides/fisiología , Proteína p53 Supresora de Tumor/genéticaRESUMEN
LCC2, an estradiol-independent tamoxifen (Tax)-resistant subline of MCF-7 human breast cancer cell line, is resistant relatively towards Tax and methotrexate (Mtx). The purpose of the present study is to evaluate the role of p53 in determining this resistance. While MCF-7 is sensitive to and undergoes apoptosis, as determined by propidium iodide stain, by Tax and Mtx, LCC2 is resistant to apoptosis induction by these agents. Both cell lines undergo apoptosis and are sensitive equally to doxorubicin (Adr). p53 cDNA of both sublines was evaluated by polymerase chain reaction (PCR) amplification and sequencing and was found to be of wild-type. p53 mRNA, as well as protein, are elevated markedly in LCC2 as compared to MCF-7 cells. p53 expression was increased by estradiol and Adr, not changed by Mtx, and decreased by Tax and estradiol-deprivation in both sublines. p53 modulation by the various agents, in both sublines, was evaluated by cytochemical staining and subcellular fractionation. This analysis showed that p53 is localized mainly in the nuclear fraction in MCF-7 cells, and in the cytoplasmatic fraction in LCC2 cells. Doxorubicin induced apoptosis in MCF-7 cells along with increase in its nuclear fraction. In contrast, LCC2 underwent apoptosis by Adr despite its cytoplasmatic sequestration. These experiments demonstrate that p53 is sequestered to cytoplasm in the estrogen-independent, Tax-resistant LCC2 cells. However, the differences in apoptotic rate between MCF-7 and LCC2 cells do not seem to be dependent on p53. The LCC2 cell line may serve as a useful model for the study of the mechanism of cytoplasmatic sequestration of wild type (wt) p53, its physiologic consequences, and its relation to estrogen-independence or Tax resistance of breast cancer cells.
Asunto(s)
Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/metabolismo , Estradiol/farmacología , Neoplasias Hormono-Dependientes/metabolismo , Células Tumorales Cultivadas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis/efectos de los fármacos , Northern Blotting , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Cartilla de ADN/química , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Femenino , Humanos , Técnicas para Inmunoenzimas , Metotrexato/farmacología , Neoplasias Hormono-Dependientes/tratamiento farmacológico , Propidio , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fracciones Subcelulares , Tamoxifeno/farmacología , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The ability to suppress wild type p53-independent apoptosis may play an important role in the oncogenicity of p53 mutant proteins. However, structural elements necessary for this activity are unknown. Furthermore, it is unclear whether this mutant p53 mediated inhibition is specific to the apoptotic pathway or a more general suppression of the cellular response to stress. We observed that an unmodified C-terminus was required for the suppression of apoptosis by the p53 135(Ala to Val) oncogenic p53 mutant. It was also required for the novel activity of G2 arrest suppression, the predominant response at low levels of genotoxic stress. These observations are consistent with a model whereby mutant p53 suppressive activity is not specific to the apoptotic pathway, but rather increases the threshold of genotoxic stress needed for a DNA damage response to occur. Furthermore, these observations indicate that it may be possible to selectively kill mutant p53 expressing cells based on the lower sensitivity of their growth arrest response.
Asunto(s)
Apoptosis , Mutación , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/efectos de los fármacos , División Celular , Cisplatino/farmacología , Daño del ADN , Etopósido/farmacología , Fase G2 , Humanos , Ratones , Proteína p53 Supresora de Tumor/químicaRESUMEN
The present study examined whether the ability of mutant p53 to block apoptosis depended on its transcriptional activity. A core domain mutant p53 (143 Val to Ala), in which two N-terminal residues (22 and 23) essential for transactivation were also mutated (Leu to Glu and Trp to Ser, respectively), was examined. While p53 containing only the core mutation efficiently interfered with drug-induced apoptosis, further modification at the N-terminus abolished this blocking activity. Furthermore, expression of c-myc, a suggested target for core mutant p53 transactivation, was elevated in the core mutant p53-expressing cells, but was abolished in the presence of the transcription-deficient p53 core mutant. In addition, wild-type p53, mutated in the N-terminus (residues 22 and 23), was unable to induce apoptosis by itself. Nevertheless, it synergized with drugs in the induction of apoptosis. This suggests that the integrity of the N-terminus is essential for both the activity of wild-type p53 in apoptosis and for mutant p53-mediated block of drug-induced apoptosis. This supports the notion that core p53 mutants act via a gain of function mechanism.
Asunto(s)
Apoptosis , Transactivadores/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Alanina/genética , Amanitinas/farmacología , Línea Celular , Cisplatino/farmacología , Dactinomicina/farmacología , Doxorrubicina/farmacología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Sustancias Intercalantes/farmacología , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa II/antagonistas & inhibidores , Relación Estructura-Actividad , Transactivadores/genética , Proteína p53 Supresora de Tumor/genética , Valina/genéticaRESUMEN
Mdm-2 plays a central role in the regulation of p53 protein level and activity. Although the interaction of mdm-2 and p53 occurs through the N-terminus of the p53 protein, our present data suggest that the C' terminus plays an important role in the regulation of the p53/mdm-2 loop. Comparative analysis of the murine regularly spliced form of p53 (RSp53) and a physiological C-terminally modified p53 protein, which results from alternative splicing of the p53 mRNA (ASp53), indicated that the two isoforms behave differently in the p53/mdm-2 loop. We found that ASp53 can preferentially induce higher levels of the mdm-2 protein, compared with RSp53. Although the transactivation capacity of both forms is inhibited by mdm-2, only RSp53 is directed to proteolytic degradation by mdm-2, while ASp53 is relatively resistant. We present evidence that suggests that ASp53 protein levels determine the biological activities mediated by RSp53, such as the induction of apoptosis, through the mdm-2/p53 regulatory loop. We suggest, therefore, a new mechanism for the regulation of p53, and show that alteration of the p53 extreme C' terminus can significantly change the transcription activity and the resistance to degradation properties of the p53 protein.
Asunto(s)
Proteínas Nucleares , Proteínas Proto-Oncogénicas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Empalme Alternativo , Animales , Ratones , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-mdm2 , Transcripción Genética , Proteína p53 Supresora de Tumor/químicaRESUMEN
The cellular function of p53 is complex. It is well known that p53 plays a key role in cellular response to DNA damage. Moreover, p53 was implicated in cellular senescence, and it was demonstrated that p53 undergoes modification in senescent cells. However, it is not known how these modifications affect the ability of senescent cells to respond to DNA damage. To address this question, we studied the responses of cultured young and old normal diploid human fibroblasts to a variety of genotoxic stresses. Young fibroblasts were able to undergo p53-dependent and p53-independent apoptosis. In contrast, senescent fibroblasts were unable to undergo p53-dependent apoptosis, whereas p53-independent apoptosis was only slightly reduced. Interestingly, instead of undergoing p53-dependent apoptosis, senescent fibroblasts underwent necrosis. Furthermore, we found that old cells were unable to stabilize p53 in response to DNA damage. Exogenous expression or stabilization of p53 with proteasome inhibitors in old fibroblasts restored their ability to undergo apoptosis. Our results suggest that stabilization of p53 in response to DNA damage is impaired in old fibroblasts, resulting in induction of necrosis. The role of this phenomenon in normal aging and anticancer therapy is discussed.
Asunto(s)
Senescencia Celular , Daño del ADN , Fibroblastos/metabolismo , Fibroblastos/patología , Proteína p53 Supresora de Tumor/metabolismo , Naranja de Acridina/farmacología , Antibióticos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Apoptosis , Western Blotting , Separación Celular , Células Cultivadas , Cisplatino/farmacología , Cisteína Endopeptidasas , Dactinomicina/farmacología , Electroforesis en Gel de Poliacrilamida , Etopósido/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , Citometría de Flujo , Colorantes Fluorescentes/farmacología , Humanos , Modelos Biológicos , Complejos Multienzimáticos/antagonistas & inhibidores , Necrosis , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Complejo de la Endopetidasa Proteasomal , Factores de Tiempo , Transfección , Proteína p53 Supresora de Tumor/biosíntesis , Rayos UltravioletaRESUMEN
To elucidate the nature of the cross-talk between the p53 protein and the DNA repair machinery, we have investigated the relationship between the two throughout the cell cycle. Base excision repair (BER) was analyzed in cell cycle phase-enriched populations of lymphoid cells expressing wild-type p53. Our study yielded the following novel findings: (a) BER exhibited two distinct peaks of activity, one associated with the G0-G1 checkpoint and the second with the G2-M checkpoint; (b) although the overall BER activity was reduced after exposure of cells to 400R, there was an augmentation of the G0-G1-associated BER activity and a reduction in the G2-M-associated BER activity; and (c) modulations in these patterns of BER after genotoxic stress were found to be p53 regulated. p53 protein levels induced after gamma-irradiation were distributed evenly in the various cell cycle populations (analyzed by the PAb-248 anti-p53 monoclonal antibody). However, both the dephosphorylation of serine 376 of p53 (contained in the PAb-421 epitope) and the specific DNA binding activity, as well as apoptosis, were enhanced toward the G2-M populations. Furthermore, inactivation of wild-type p53, mediated by mutant p53 expression, abolished the alterations in the BER pattern and showed no induction of a G2-M-associated apoptosis after gamma-irradiation. These results suggest that after genotoxic stress, stabilized p53 enhances the G0-G1-associated BER activity, whereas it predominantly reduces BER activity at the G2-M-enriched populations and instead induces apoptosis. After genotoxic stress, p53 functions as a modulator that determines the pattern of BER activity and apoptosis in a cell cycle-specific manner.
Asunto(s)
Ciclo Celular/fisiología , Reparación del ADN/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Apoptosis/genética , Apoptosis/efectos de la radiación , Linfocitos B/citología , Linfocitos B/efectos de la radiación , Ciclo Celular/efectos de la radiación , División Celular/genética , División Celular/efectos de la radiación , Línea Celular/citología , Línea Celular/efectos de la radiación , Separación Celular , Centrifugación , ADN/biosíntesis , Daño del ADN , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Rayos gamma , Interfase/fisiología , Interfase/efectos de la radiación , Ratones , Mitosis/fisiología , Mitosis/efectos de la radiación , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Genome instability is a primary factor leading to the activation of the p53 tumor suppressor protein. Telomeric repeat (TR) sequences are also responsible for genome integrity. By capping the termini of the chromosomes, TRs prevent them undergoing nucleolytic degradation, ligation or chromosome fusion. Interestingly, telomere shortening was suggested to activate p53, which in turn may cause primary cells to senesce. In order to elucidate the nature of a possible cross talk between the two, we introduced into cells TRs of defined length and investigated their effect on p53 activation and subsequent cellular response. We found that the introduction of a TR into cells leads to stabilization of the p53 protein. This stabilization was specific to TRs and was not observed in response to exposure of cells to plasmids containing non-TR sequences. p53 stabilization requires the presence of an intact p53 oligomerization domain. TR-activated p53 exhibited enhanced transcriptional activity. Eventually, TRs induced p53-dependent growth suppression, measured as a reduction in colony formation.
Asunto(s)
Secuencias Repetitivas de Ácidos Nucleicos/genética , Telómero/genética , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , Sitios de Unión/genética , División Celular/genética , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Plásmidos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Activación Transcripcional , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genéticaRESUMEN
It is now well accepted that the p53 C-terminus plays a central role in controlling the activity of the wild-type molecule. In our previous studies, we observed that a C-terminally altered p53 protein (p53AS), generated by an alternative spliced p53 mRNA, induces an attenuated p53-dependent apoptosis, compared to that induced by the regularly spliced form (p53RS). In the present study we analysed the interrelationships between these two physiological variants of wild-type p53, and found that in cells co-expressing both forms, in contrast to the expected additive effect on the induction of apoptosis, p53AS inhibits apoptosis induced by p53RS. This inhibitory effect is specific for p53-dependent apoptosis and was not evident in a p53-independent apoptotic pathway induced by growth factor deprivation. Furthermore, the expression of p53AS in transiently transfected cells caused both inhibition of apoptosis and inhibition of the p53RS-dependent transactivation of a number of p53 target genes. These results suggest that expression of an alternatively spliced p53 form may serve as an additional level in controlling the complexity of p53 function by the C-terminal domain.
Asunto(s)
Empalme Alternativo , Apoptosis , Proteína p53 Supresora de Tumor/metabolismo , Animales , Ciclina G , Ciclina G1 , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/genética , Regulación de la Expresión Génica , Humanos , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transcripción Genética , Transfección , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/genética , Proteína X Asociada a bcl-2RESUMEN
Previously, it was implicated that p53 plays a role in spermatogenesis. Here we report that p53 knockout mice exhibit significantly less mature motile spermatozoa than their p53(+/+) counterparts. To better understand the role of p53 in spermatogenesis, we analyzed the response of spermatogenic cells to DNA insult during prophase. It was found that although low-level gamma-irradiation activated a p53-dependent premeiotic delay, higher levels of gamma-irradiation induced a p53-independent apoptosis during meiosis. Furthermore, p53 knockout mice exhibited reduced in vivo levels of unscheduled DNA synthesis, indicative of compromised DNA repair. Thus, p53 provides another level of stringency in addition to other spermatogenic "quality control" mechanisms.
Asunto(s)
Daño del ADN , Reparación del ADN , Espermatogénesis/genética , Proteína p53 Supresora de Tumor/fisiología , Animales , Replicación del ADN , Citometría de Flujo , Genotipo , Masculino , Ratones , Ratones Endogámicos C57BL , Motilidad Espermática/genética , Espermatogénesis/efectos de la radiaciónRESUMEN
The present study shows that DNA damage induces different patterns of p53-dependent and p53-independent apoptosis in epithelial cells of various organs of adult mice. Genotoxic stress induced a biphasic apoptotic response in the small intestine and tongue. While the first immediate apoptotic wave was p53-dependent, the second was slower in rate and was p53-independent. Under the same experimental conditions a single rapid, but a more extended, p53-independent response was evident in the skin of the tail. Indeed, exposure of p53+/+ mice to 400 R induced in epithelium of the small intestine and tongue an immediate rapid response that was followed by a second delayed p53-independent apoptotic wave. p53-/- mice exhibited in these organs the second wave only. However, epithelium of the tail derived from the same mice showed a single rapid apoptotic response that lasted much longer than the p53-dependent response and was similar in the p53-/- and the p53+/+ mice. Variations in apoptotic patterns observed in epithelial cells derived of the different tissues may point to differences in the physiological pathways expressed.
Asunto(s)
Apoptosis/genética , Daño del ADN , Genes p53 , Animales , Apoptosis/efectos de la radiación , Secuencia de Bases , ADN/genética , ADN/efectos de la radiación , Cartilla de ADN/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Epiteliales/efectos de la radiación , Femenino , Rayos gamma , Intestino Delgado/citología , Intestino Delgado/metabolismo , Intestino Delgado/efectos de la radiación , Ratones , Ratones Noqueados , Especificidad de Órganos , Piel/citología , Piel/metabolismo , Piel/efectos de la radiaciónRESUMEN
In order to identify the alternative pathways which may substitute for the p53 function during embryogenesis, we have focused our studies on p53 -/- normally developing mouse embryos that survived a genotoxic stress. We assumed that under these conditions p53-independent pathways, which physiologically control genomic stability, are enhanced. We found that while p53 +/+ mouse embryos elicited, as expected, a p53-dependent apoptosis, p53-/- normally developing mice exhibited an accentuated p53-independent apoptotic response. The p53-dependent apoptosis detected in p53+/+ embryos, was an immediate reaction mostly detected in the brain, whereas the p53-independent apoptosis was a delayed reaction with a prominent pattern observed in epithelial cells of most organs in the p53-deficient mice only. These results suggest that in the absence of p53-dependent apoptosis, which is a fast response to damaged DNA, p53-independent apoptotic pathways, with slower kinetics, are turned on to secure genome stability.