RESUMEN

Oligonucleotide/oligosaccharide-binding (OB) domain-containing proteins have been identified as critical for telomere maintenance, DNA repair, transcription and other DNA metabolism processes. Protection of telomere 1 (POT1), a telomere binding protein, has an OB domain like single-strand binding protein (SSB1). In this issue of Oncogene, Gu et al. present evidence that POT1, like SSB1, is required to maintain genomic stability. This work, in conjunction with results from previous investigators, highlights the importance of POT1 in telomere metabolism. Inactivation of POT1 telomere protective functions in mouse models lacking p53 expression in the breast epithelium unleashes a torrent of DNA damage responses (DDRs) at the telomeres, culminating in karyotypic alterations with massive arrays of telomere fusions. Therefore, POT1 is not only required to promote telomere homeostasis, but also plays an essential role in maintaining a stable genome.

Asunto(s)

Proteínas de Unión a Telómeros/química , Telómero , Animales , Inestabilidad Genómica , Homeostasis del Telómero

RESUMEN

MOF (males absent on the first) was initially discovered as a dosage compensation factor that regulates the epigenetic acetylation of histone H4 lysine 16. In this issue, Sheikh et al. demonstrate that MOF expression is not required for normal kidney tissue function but is required for maintaining transcriptional regulation under conditions of stress. This work along with results from previous investigators highlights the importance of the cell lineage-chromatin modification interaction in determining transcriptional programs and physiological outcomes under normal and stress conditions.

Asunto(s)

Histona Acetiltransferasas/genética , Histonas , Acetilación , Regulación de la Expresión Génica , Humanos , Lisina

RESUMEN

Genomic stability is maintained by telomeres, the end terminal structures that protect chromosomes from fusion or degradation. Shortening or loss of telomeric repeats or altered telomere chromatin structure is correlated with telomere dysfunction such as chromosome end-to-end associations that could lead to genomic instability and gene amplification. The structure at the end of telomeres is such that its DNA differs from DNA double strand breaks (DSBs) to avoid nonhomologous end-joining (NHEJ), which is accomplished by forming a unique higher order nucleoprotein structure. Telomeres are attached to the nuclear matrix and have a unique chromatin structure. Whether this special structure is maintained by specific chromatin changes is yet to be thoroughly investigated. Chromatin modifications implicated in transcriptional regulation are thought to be the result of a code on the histone proteins (histone code). This code, involving phosphorylation, acetylation, methylation, ubiquitylation, and sumoylation of histones, is believed to regulate chromatin accessibility either by disrupting chromatin contacts or by recruiting non-histone proteins to chromatin. The histone code in which distinct histone tail-protein interactions promote engagement may be the deciding factor for choosing specific DSB repair pathways. Recent evidence suggests that such mechanisms are involved in DNA damage detection and repair. Altered telomere chromatin structure has been linked to defective DNA damage response (DDR), and eukaryotic cells have evolved DDR mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. Recent studies suggest that chromatin modifying factors play a critical role in the maintenance of genomic stability. This review will summarize the role of DNA damage repair proteins specifically ataxia-telangiectasia mutated (ATM) and its effectors and the telomere complex in maintaining genome stability.

Asunto(s)

Daño del ADN , Histonas/genética , Telómero/genética , Acetilación , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/ultraestructura , Proteínas de Unión al ADN/genética , Inestabilidad Genómica/genética , Histonas/metabolismo , Humanos , Metilación , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Radiación Ionizante , Telómero/efectos de la radiación , Telómero/ultraestructura , Proteínas Supresoras de Tumor/genética , Ubiquitina/genética

RESUMEN

Beyond their role in replication and chromosome end capping, telomeres are also thought to function in meiotic chromosome pairing, meiotic and mitotic chromosome segregation as well as in nuclear organization. Observations in both somatic and meiotic cells suggest that the positioning of telomeres within the nucleus is highly specific and believed to be dependent mainly on telomere interactions with the nuclear envelope either directly or through chromatin interacting proteins. Although little is known about the mechanism of telomere clustering, some studies show that it is an active process. Recent data have suggested a regulatory role for telomere chromatin structure in telomere movement. This review will summarize recent studies on telomere interactions with the nuclear matrix, telomere chromatin structure and factors that modify telomere chromatin structure as related to regulation of telomere movement.

Asunto(s)

Cromatina/genética , Segregación Cromosómica/fisiología , Meiosis/genética , Modelos Moleculares , Matriz Nuclear/metabolismo , Telómero/genética , Telómero/fisiología

RESUMEN

Meiosis pairs and segregates homologous chromosomes and thereby forms haploid germ cells to compensate the genome doubling at fertilization. Homologue pairing in many eukaryotic species depends on formation of DNA double strand breaks (DSBs) during early prophase I when telomeres begin to cluster at the nuclear periphery (bouquet stage). By fluorescence in situ hybridization criteria, we observe that mid-preleptotene and bouquet stage frequencies are altered in male mice deficient for proteins required for recombination, ubiquitin conjugation and telomere length control. The generally low frequencies of mid-preleptotene spermatocytes were significantly increased in male mice lacking recombination proteins SPO11, MEI1, MLH1, KU80, ubiquitin conjugating enzyme HR6B, and in mice with only one copy of the telomere length regulator Terf1. The bouquet stage was significantly enriched in Atm(-/-), Spo11(-/-), Mei1(m1Jcs/m1Jcs), Mlh1(-/-), Terf1(+/-) and Hr6b(-/-) spermatogenesis, but not in mice lacking recombination proteins DMC1 and HOP2, the non-homologous end-joining DNA repair factor KU80 and the ATM downstream effector GADD45a. Mice defective in spermiogenesis (Tnp1(-/-), Gmcl1(-/-), Asm(-/-)) showed wild-type mid-preleptotene and bouquet frequencies. A low frequency of bouquet spermatocytes in Spo11(-/-)Atm(-/-) spermatogenesis suggests that DSBs contribute to the Atm(-/-)-correlated bouquet stage exit defect. Insignificant changes of bouquet frequencies in mice with defects in early stages of DSB repair (Dmc1(-/-), Hop2(-/-)) suggest that there is an ATM-specific influence on bouquet stage duration. Altogether, it appears that several pathways influence telomere dynamics in mammalian meiosis.

Asunto(s)

Meiosis/genética , Mutación , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Roturas del ADN de Doble Cadena , Reparación del ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas , Esterasas/deficiencia , Esterasas/genética , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Noqueados , Profase/genética , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Recombinación Genética , Espermatocitos/citología , Espermatocitos/metabolismo , Espermatogénesis/genética , Telómero/genética , Proteínas Supresoras de Tumor/deficiencia , Proteínas Supresoras de Tumor/genética

RESUMEN

The response of xenotransplants were compared with waterbath immersion vs focal ultrasound (US) hyperthermia using tumour growth delay, immunhistochemistry and histopathology assays. Waterbath hyperthermia was performed by limb immersion. Precautions were taken to minimize total body heating by surrounding the mouse with plastic insulators. Thermometry was performed with clinical-grade, 20-gauge needle thermocouples and monitored with a Labthermics unit. Significant differences in cytotoxicity between ultrasound and waterbath treatment of tumors at 43 degrees C were observed as determined by TUNNEL assay. Conversely, contralateral (non-treated) tumours in animals treated with similar temperature demonstrated no significant differences between modalities. Western blot analysis revealed increased hsp70 induction at 43 degrees C in waterbath vs focal ultrasound hyperthermia. Comparison of tumour growth delay between tumours heated with waterbath vs ultrasound at 43 degrees C but not at 41 degrees C revealed significant differences. This is the first study comparing localized vs regional hyperthermia using the small animal ultrasound system (SAHUS) delivery system. Consistent ultrasound hyperthermia can be achieved throughout a xenotransplant. At equivalent temperature of 43 degrees C for 60?min, waterbath hyperthermia demonstrated greater local response vs ultrasound hyperthermia.

Asunto(s)

Hipertermia Inducida/métodos , Neoplasias Experimentales/terapia , Terapia por Ultrasonido , Animales , Femenino , Miembro Anterior , Células HeLa , Miembro Posterior , Humanos , Etiquetado Corte-Fin in Situ , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Carga Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

DNA damage-induced acetylation of p53 protein leads to its activation and either growth arrest or apoptosis. We show here that the protein product of the gene hSIR2(SIRT1), the human homolog of the S. cerevisiae Sir2 protein known to be involved in cell aging and in the response to DNA damage, binds and deacetylates the p53 protein with a specificity for its C-terminal Lys382 residue, modification of which has been implicated in the activation of p53 as a transcription factor. Expression of wild-type hSir2 in human cells reduces the transcriptional activity of p53. In contrast, expression of a catalytically inactive hSir2 protein potentiates p53-dependent apoptosis and radiosensitivity. We propose that hSir2 is involved in the regulation of p53 function via deacetylation.

Asunto(s)

Histona Desacetilasas/fisiología , NAD/metabolismo , Proteínas Reguladoras de Información Silente de Saccharomyces cerevisiae , Transactivadores/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis , Línea Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Daño del ADN , Relación Dosis-Respuesta en la Radiación , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Immunoblotting , Luciferasas/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Mutación , Péptidos/química , Plásmidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Sirtuina 1 , Sirtuina 2 , Sirtuinas , Transcripción Genética , Transfección

RESUMEN

Ataxia telangiectasia (AT) is a rare human autosomal recessive disorder with a wide variety of phenotypic manifestations. AT patients are cancer prone and hypersensitive to ionizing radiation. Cells derived from AT patients require higher levels of serum factors, exhibit cytoskeletal defects, and undergo premature senescence in culture. The gene responsible for AT is ATM (ataxia-telangiectasia mutated), and its product has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, and cell cycle control. Because of the homology of the human ATM gene to the TEL1 and rad3 genes of yeast, it has been suggested that mutations in ATM could lead to defective telomere maintenance. The ATM gene product influences chromosome end associations, telomere length, and telomere clustering. The defective telomere metabolism in AT cells could be due to altered interactions between the telomeres and the nuclear matrix. These interactions were studied in nuclear matrix halos before and after irradiation. Altered telomere-nuclear matrix interactions were observed in cells derived from individuals with AT. AT cells also had different nucleosomal periodicity in their telomeres from normal cells. Both telomere-nuclear matrix interactions and nucleosomal periodicity were altered by treatment of primary AT fibroblasts with ionizing radiation. This effect was not observed in cells derived from normal individuals. A link was also found between altered telomere-nuclear matrix interactions, aberrant telomere clustering, and gonadal atrophy. The telomere defect was not corrected by the ectopic expression of the catalytic subunit of telomerase (TERT). Since alteration of the yeast telomere chromatin structure is known to influence gene expression, we compared expressed sequence tags (ESTs) of Atm-null mouse cells and normal mouse cells. Several ESTs were found to be aberrantly expressed in Atm-null mouse cells. This paper summarizes our recent publications and presents some new data on the influence of ATM on telomere metabolism.

Asunto(s)

Proteínas Serina-Treonina Quinasas/fisiología , Telómero/fisiología , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Células Cultivadas , Cromatina/metabolismo , Aberraciones Cromosómicas , Cromosomas/metabolismo , Proteínas de Unión al ADN , Expresión Génica , Humanos , Matriz Nuclear/metabolismo , Nucleosomas/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/efectos de la radiación , Radiación Ionizante , Telómero/química , Proteínas Supresoras de Tumor

RESUMEN

The study of Ataxia-telangiectasia (A-T) has benefited significantly from mouse models with knockout mutations for the Atm (A-T mutation) locus. While these models have proven useful for in vivo studies, cell cultures from Atm null embryos have been reported to grow poorly and then senesce. In this study, we initiated primary cultures from adult ears and kidneys of Atm homozygous mice and found that these cultures immortalized readily without loss of sensitivity to ionizing radiation and other Atm related cell cycle defects. A mutational analysis for loss of expression of an autosomal locus showed that ionizing radiation had a mutagenic effect. Interestingly, some spontaneous mutants exhibited a mutational pattern that is characteristic of oxidative mutagenesis. This result is consistent with chronic oxidative stress in Atm null cells. In total, the results demonstrate that permanent cell lines can be established from the tissues of adult mice homozygous for Atm and that these cell lines will exhibit expected and novel consequences of this deficiency.

Asunto(s)

Ataxia Telangiectasia/genética , Línea Celular Transformada , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/genética , Radiación Ionizante , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/efectos de la radiación , Proteínas de Ciclo Celular , Supervivencia Celular/efectos de la radiación , Aberraciones Cromosómicas , Proteínas de Unión al ADN , Pérdida de Heterocigocidad/efectos de la radiación , Metafase/efectos de la radiación , Ratones , Ratones Noqueados , Mutagénesis , Mutación , Tolerancia a Radiación , Proteínas Supresoras de Tumor

RESUMEN

Ataxia-telangiectasia (A-T) is an autosomal recessive disease characterized by progressive cerebellar degeneration, immunodeficiencies, genomic instability and gonadal atrophy. A-T patients are hypersensitive to ionizing radiation and have an elevated cancer risk. Cells derived from A-T patients require higher levels of serum factors, exhibit cytoskeletal defects and undergo premature senescence in culture. We show here that expression of the catalytic subunit of telomerase (hTERT) in primary A-T patient fibroblasts can rescue the premature senescence phenotype. Ectopic expression of hTERT does not rescue the radiosensitivity or the telomere fusions in A-T fibroblasts. The hTERT+AT cells also retain the characteristic defects in cell-cycle checkpoints, and show increased chromosome damage before and after ionizing radiation. Although A-T patients have an increased susceptibility to cancer, the expression of hTERT in A-T fibroblasts does not stimulate malignant transformation. These immortalized A-T cells provide a more stable cell system to investigate the molecular mechanisms underlying the cellular phenotypes of Ataxia-telangiectasia.

Asunto(s)

Ataxia Telangiectasia/patología , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , ARN , Telomerasa/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Pruebas de Carcinogenicidad , Ciclo Celular/efectos de la radiación , Línea Celular Transformada , Senescencia Celular , Cromosomas Humanos/genética , Cromosomas Humanos/efectos de la radiación , Daño del ADN/efectos de la radiación , Proteínas de Unión al ADN , Fibroblastos/patología , Fibroblastos/virología , Humanos , Masculino , Ratones , Ratones Desnudos , Tolerancia a Radiación , Radiación Ionizante , Valores de Referencia , Retroviridae/genética , Telomerasa/genética , Telómero/genética

RESUMEN

Cells from patients with the genetic disorder ataxia-telangiectasia (A-T) are hypersensitive to ionizing radiation and radiomimetic agents, both of which generate reactive oxygen species capable of causing oxidative damage to DNA and other macromolecules. We describe in A-T cells constitutive activation of pathways that normally respond to genotoxic stress. Basal levels of p53 and p21(WAF1/CIP1), phosphorylation on serine 15 of p53, and the Tyr15-phosphorylated form of cdc2 are chronically elevated in these cells. Treatment of A-T cells with the antioxidant alpha-lipoic acid significantly reduced the levels of these proteins, pointing to the involvement of reactive oxygen species in their chronic activation. These findings suggest that the absence of functional ATM results in a mild but continuous state of oxidative stress, which could account for several features of the pleiotropic phenotype of A-T.

Asunto(s)

Antioxidantes/farmacología , Ataxia Telangiectasia/tratamiento farmacológico , Ataxia Telangiectasia/metabolismo , Ácido Tióctico/farmacología , Ataxia Telangiectasia/patología , Proteína Quinasa CDC2/efectos de los fármacos , Proteína Quinasa CDC2/metabolismo , Estudios de Casos y Controles , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/efectos de los fármacos , Ciclinas/metabolismo , Cicloheximida/farmacología , Daño del ADN/efectos de los fármacos , Humanos , Estrés Oxidativo , Fosforilación , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Tirosina/metabolismo

RESUMEN

Treatment failure after radiation therapy of prostate cancer (PC) could be a significant problem. Our objective is to design genetic radiosensitizing strategies for the treatment of PC. Cells from individuals with the genetic disorder ataxia telangiectasia (AT) are hypersensitive to ionizing radiation. Therefore, we examined whether attenuation of the AT gene product, AT mutated (ATM), in PC cells could result in an increased intrinsic radiosensitivity. A p53-mutant PC cell line, PC-3 was infected with adenoviral vectors, expressing antisense ATM RNA to various domains of the ATM gene. Immunoblot analyses of cellular extracts from antisense ATM-transfected PC-3 cells showed attenuated expression of the ATM protein within 2 days of viral infection. Compared with cells infected with an adeno-beta-galactosidase vector, antisense ATM-transfected PC-3 cells showed aberrant control of S-phase cell-cycle checkpoints after exposure to ionizing radiation. Under these conditions, the intrinsic radiosensitivity of the PC-3 cells was enhanced. Consequently antisense ATM gene therapy could serve as a paradigm for strategies that target the cellular survival mechanisms of an irradiated tumor cell and may provide therapeutic benefit to patients undergoing radiation therapy for PC.

Asunto(s)

Adenoviridae/genética , Neoplasias de la Próstata/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN sin Sentido/genética , Tolerancia a Radiación/genética , Transfección , Células Tumorales Cultivadas/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular , Proteínas de Ciclo Celular , División Celular , Supervivencia Celular/efectos de la radiación , Daño del ADN/efectos de la radiación , Cartilla de ADN/química , Proteínas de Unión al ADN , Relación Dosis-Respuesta en la Radiación , Citometría de Flujo , Vectores Genéticos , Humanos , Immunoblotting , Masculino , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Serina-Treonina Quinasas/genética , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor

RESUMEN

Telomeres are complexes of repetitive DNA sequences and proteins constituting the ends of linear eukaryotic chromosomes. While these structures are thought to be associated with the nuclear matrix, they appear to be released from this matrix at the time when the cells exit from G(2) and enter M phase. Checkpoints maintain the order and fidelity of the eukaryotic cell cycle, and defects in checkpoints contribute to genetic instability and cancer. The 14-3-3sigma gene has been reported to be a checkpoint control gene, since it promotes G(2) arrest following DNA damage. Here we demonstrate that inactivation of this gene influences genome integrity and cell survival. Analyses of chromosomes at metaphase showed frequent losses of telomeric repeat sequences, enhanced frequencies of chromosome end-to-end associations, and terminal nonreciprocal translocations in 14-3-3sigma(-/-) cells. These phenotypes correlated with a reduction in the amount of G-strand overhangs at the telomeres and an altered nuclear matrix association of telomeres in these cells. Since the p53-mediated G(1) checkpoint is operative in these cells, the chromosomal aberrations observed occurred preferentially in G(2) after irradiation with gamma rays, corroborating the role of the 14-3-3sigma protein in G(2)/M progression. The results also indicate that even in untreated cycling cells, occasional chromosomal breaks or telomere-telomere fusions trigger a G(2) checkpoint arrest followed by repair of these aberrant chromosome structures before entering M phase. Since 14-3-3sigma(-/-) cells are defective in maintaining G(2) arrest, they enter M phase without repair of the aberrant chromosome structures and undergo cell death during mitosis. Thus, our studies provide evidence for the correlation among a dysfunctional G(2)/M checkpoint control, genomic instability, and loss of telomeres in mammalian cells.

Asunto(s)

Biomarcadores de Tumor , Fragilidad Cromosómica/genética , Exonucleasas , Eliminación de Gen , Proteínas de Neoplasias , Proteínas/metabolismo , Telómero/genética , Telómero/efectos de la radiación , Proteínas 14-3-3 , División Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Cromatina/genética , Cromatina/efectos de la radiación , Bandeo Cromosómico , Rotura Cromosómica/genética , Relación Dosis-Respuesta en la Radiación , Exorribonucleasas , Fase G1 , Fase G2 , Rayos gamma , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Índice Mitótico , Matriz Nuclear/metabolismo , Proteínas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Secuencias Repetitivas de Ácidos Nucleicos/efectos de la radiación , Cromosomas en Anillo , Telómero/metabolismo , Translocación Genética/genética , Translocación Genética/efectos de la radiación , Células Tumorales Cultivadas

RESUMEN

The ataxia telangiectasia mutant (ATM) protein is an intrinsic part of the cell cycle machinery that surveys genomic integrity and responses to genotoxic insult. Individuals with ataxia telangiectasia as well as Atm(-/-) mice are predisposed to cancer and are infertile due to spermatogenesis disruption during first meiotic prophase. Atm(-/-) spermatocytes frequently display aberrant synapsis and clustered telomeres (bouquet topology). Here, we used telomere fluorescent in situ hybridization and immunofluorescence (IF) staining of SCP3 and testes-specific histone H1 (H1t) to spermatocytes of Atm- and Atm-p53-deficient mice and investigated whether gonadal atrophy in Atm-null mice is associated with stalling of telomere motility in meiotic prophase. SCP3-H1t IF revealed that most Atm(-/-) p53(-/-) spermatocytes degenerated during late zygotene, while a few progressed to pachytene and diplotene and some even beyond metaphase II, as indicated by the presence of a few round spermatids. In Atm(-/-) p53(-/-) meiosis, the frequency of spermatocytes I with bouquet topology was elevated 72-fold. Bouquet spermatocytes with clustered telomeres were generally void of H1t signals, while mid-late pachytene and diplotene Atm(-/-) p53(-/-) spermatocytes displayed expression of H1t and showed telomeres dispersed over the nuclear periphery. Thus, it appears that meiotic telomere movements occur independently of ATM signaling. Atm inactivation more likely leads to accumulation of spermatocytes I with bouquet topology by slowing progression through initial stages of first meiotic prophase and an ensuing arrest and demise of spermatocytes I. Sertoli cells (SECs), which contribute to faithful spermatogenesis, in the Atm mutants were found to frequently display numerous heterochromatin and telomere clusters-a nuclear topology which resembles that of immature SECs. However, Atm(-/-) SECs exhibited a mature vimentin and cytokeratin 8 intermediate filament expression signature. Upon IF with ATM antibodies, we observed ATM signals throughout the nuclei of human and mouse SECs, spermatocytes I, and haploid round spermatids. ATM but not H1t was absent from elongating spermatid nuclei. Thus, ATM appears to be removed from spermatid nuclei prior to the occurrence of DNA nicks which emanate as a consequence of nucleoprotamine formation.

Asunto(s)

Núcleo Celular/metabolismo , Eliminación de Gen , Genes p53/fisiología , Meiosis , Proteínas Serina-Treonina Quinasas/fisiología , Células de Sertoli/citología , Telómero/metabolismo , Animales , Ataxia Telangiectasia/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Núcleo Celular/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas de Unión al ADN , Colorantes Fluorescentes , Genes p53/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Inmunohistoquímica , Hibridación Fluorescente in Situ , Masculino , Ratones , Ratones Noqueados , Matriz Nuclear/genética , Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Células de Sertoli/metabolismo , Espermatocitos/citología , Espermatocitos/metabolismo , Telómero/genética , Proteínas Supresoras de Tumor , Vimentina/metabolismo

RESUMEN

The ATM (ataxia telangiectasia mutated) gene product has been implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, and cell cycle control. The human ATM protein shows similarity to several yeast and mammalian proteins involved in meiotic recombination and cell cycle progression. Because of the homology of the human ATM gene to the TEL1 and rad3 genes of yeast, it has been suggested that mutations in ATM could lead to defective telomere maintenance. Recently, we have shown that the ATM gene product, which is defective in the cancer-prone disorder ataxia telangiectasia (AT), influences chromosome end associations and telomere length. A possible hypothesis explaining these results is that the defective telomere metabolism in AT cells is due to altered interactions between the telomeres and the nuclear matrix. These interactions were examined in nuclear matrix halos prior to and after irradiation. A difference was observed in the ratio of soluble and matrix-associated telomeric DNA between cells derived from AT and normal individuals. Treatment with ionizing radiation affected the ratio of soluble and matrix-associated telomeric DNA only in the AT cells. To test the hypothesis that the ATM gene product is involved in interactions between telomeres and the nuclear matrix, such interactions were examined in human cells expressing either a dominant-negative effect or complementation of the ATM gene. The phenotype of RKO colorectal tumor cells expressing ATM fragments containing a leucine zipper motif mimics the altered interactions of telomere and nuclear matrix seen in AT cells. Fibroblasts from AT individuals transfected with a wild-type ATM gene had corrected telomere-nuclear matrix interactions. In experiments designed to determine whether there is a link between the altered telomere-nuclear matrix interactions and defective telomere movement and clustering, a significant difference was observed in the ratio of soluble compared to matrix-associated telomeric DNA sequences in meiocytes of Atm(-/-) and control mice. These results suggest that the ATM gene influences the interactions between telomeres and the nuclear matrix and that alterations in telomere chromatin could be at least partly responsible for the pleiotropic phenotypes of the ATM gene. This paper summarizes our recent publications on the influence of inactivation of ATM on the interaction of telomeres with nuclear matrix in somatic and germ cells.

Asunto(s)

Ataxia Telangiectasia/genética , Mutación , Proteínas Serina-Treonina Quinasas/genética , Telómero/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Línea Celular , Cromosomas/química , ADN/química , Proteínas de Unión al ADN/análisis , Fibroblastos , Rayos gamma , Humanos , Masculino , Ratones , Matriz Nuclear/química , Matriz Nuclear/metabolismo , Matriz Nuclear/efectos de la radiación , Proteínas Serina-Treonina Quinasas/química , Transducción de Señal , Espermatozoides/química , Espermatozoides/metabolismo , Telómero/química , Telómero/efectos de la radiación , Proteínas Supresoras de Tumor

RESUMEN

Ionizing radiation (IR) treatment results in activation of the nonreceptor tyrosine kinase c-Abl because of phosphorylation by ATM. In vitro evidence indicates that DNA-dependent protein kinase (DNA-PK) can also phosphorylate and thus potentially activate Abl kinase activity in response to IR exposure. To unravel the role of ATM and DNA-PK in the activation of Abl, we assayed Abl, ATM, and DNA-PK activity in ATM- and DNA-PKcs-deficient cells after irradiation. Our results show that despite the presence of higher than normal levels of DNA-PK kinase activity, c-Abl fails to become activated after IR exposure in ATM-deficient cells. Conversely, normal activation of both ATM and c-Abl occurs in DNA-PKcs-deficient cells, indicating that ATM but not DNA-PK is required for activation of Abl in response to IR treatment. Moreover, activation of Abl kinase activity by IR correlates well with activation of ATM activity in all phases of the cell cycle. These results indicate that ATM is primarily responsible for activation of Abl in response to IR exposure in a cell cycle-independent fashion. Examination of DNA-PK activity in response to IR treatment in Abl-deficient cells expressing mutant forms of Abl or in normal cells exposed to an inhibitor of Abl suggests an in vivo role for Abl in the down-regulation of DNA-PK activity. Collectively, these results suggest a convergence of the ATM and DNA-PK pathways in the cellular response to IR through c-Abl kinase.

Asunto(s)

Ataxia Telangiectasia/metabolismo , Proteínas de Unión al ADN , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/efectos de la radiación , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Ciclo Celular/fisiología , Proteínas de Ciclo Celular , Daño del ADN , Proteína Quinasa Activada por ADN , Activación Enzimática , Rayos gamma , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Proteínas Nucleares , Fosforilación , Proteínas Supresoras de Tumor

RESUMEN

BACKGROUND: Telomeres consist of repetitive (TTAGGG) DNA sequences that are maintained by the multisubunit telomerase ribonucleoprotein. Telomerase consists of an RNA, which serves as template for the sequence tracts, and a catalytic subunit that functions in reverse transcription of the RNA template. Cloning and characterization of the human catalytic subunit of telomerase (hTERT) has supported a role in cell transformation. How telomerase activity is regulated, however, is largely unknown. RESULTS: We show here that hTERT associates directly with the c-Abl protein tyrosine kinase. We also found that c-Abl phosphorylates hTERT and inhibits hTERT activity. Moreover, our findings demonstrate that exposure of cells to ionizing radiation induces tyrosine phosphorylation of hTERT by a c-Abl-dependent mechanism. The functional significance of the c-Abl-hTERT interaction is supported by the demonstration that cells deficient in c-Abl show telomere lengthening. CONCLUSIONS: The ubiquitously expressed c-Abl tyrosine kinase is activated by DNA double-strand breaks. Our finding of telomere lengthening in c-Abl-deficient cells and the functional interactions between c-Abl and hTERT support a role for c-Abl in the regulation of telomerase function.

Asunto(s)

Regulación Enzimológica de la Expresión Génica , Proteínas Proto-Oncogénicas c-abl/metabolismo , ARN , Telomerasa/genética , Telomerasa/metabolismo , Dominio Catalítico , Células Cultivadas , Proteínas de Unión al ADN , Fibroblastos , Humanos , Immunoblotting , Fosforilación , Pruebas de Precipitina , Proteínas Proto-Oncogénicas c-abl/genética , Telómero/fisiología , Transfección

RESUMEN

Telomerase, a specialized reverse transcriptase (RT) linked to cell immortalization and cancer, has been thought not to be expressed in postmitotic cells. We now report that telomerase activity and its essential catalytic subunit, telomerase reverse transcriptase (TERT), are expressed in neurons in the brains of rodents during embryonic and early postnatal development, and are subsequently downregulated. Suppression of TERT expression in cultured embryonic hippocampal neurons increases their vulnerability to apoptosis and excitotoxicity. Overexpression of TERT in PC12 cells suppresses apoptosis induced by trophic factor withdrawal. TERT exerts its anti-apoptotic action at an early stage of the cell death process prior to mitochondrial dysfunction and caspase activation. TERT may serve a neuron survival-promoting function in the developing brain, and downregulation of TERT in the adult brain may contribute to increased neuronal vulnerability in various age-related neurodegenerative disorders.

Asunto(s)

Encéfalo/enzimología , Regulación del Desarrollo de la Expresión Génica , Neuronas/citología , Neuronas/enzimología , ADN Polimerasa Dirigida por ARN/genética , Telomerasa/genética , Telomerasa/metabolismo , Envejecimiento , Animales , Apoptosis , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Caspasas/metabolismo , Dominio Catalítico , Supervivencia Celular , Células Cultivadas , Desarrollo Embrionario y Fetal , Regulación Enzimológica de la Expresión Génica , Sustancias Macromoleculares , Ratones , Mitocondrias/metabolismo , Ratas , Telomerasa/química

RESUMEN

Atm, the gene mutated in ataxia-telangiectasia (AT) patients, is an essential component of the signal transduction pathway that responds to DNA damage due to ionizing radiation (IR). We attenuated ATM protein expression in human glioblastoma cells by expressing antisense RNA to a functional domain of the atm gene. While ATM expression decreased, constitutive expression of p53 and p21 increased. Irradiated ATM-attenuated cells failed to induce p53, demonstrated radioresistant DNA synthesis, and increased radiosensitivity. Antisense-ATM gene therapy in conjunction with radiation therapy may provide a novel strategy for the treatment of cancer.

Asunto(s)

Terapia Genética/métodos , Glioblastoma/genética , Proteínas Serina-Treonina Quinasas/genética , ARN sin Sentido/genética , Tolerancia a Radiación/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Supervivencia Celular/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , ADN de Neoplasias/biosíntesis , Proteínas de Unión al ADN , Glioblastoma/metabolismo , Glioblastoma/patología , Humanos , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Transfección , Células Tumorales Cultivadas/efectos de la radiación , Ensayo de Tumor de Célula Madre , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor

RESUMEN

Expression of 14-3-3 final sigma (final sigma) is induced in response to DNA damage, and causes cells to arrest in G(2). By SAGE (serial analysis of gene expression) analysis, we identified final sigma as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, final sigma mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at final sigma such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the final sigma gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of final sigma is functionally important, because treatment of final sigma-non-expressing breast cancer cell lines with the drug 5-aza-2'-deoxycytidine resulted in demethylation of the gene and synthesis of final sigma mRNA. Breast cancer cells lacking final sigma expression showed increased number of chromosomal breaks and gaps when exposed to gamma-irradiation. Therefore, it is possible that loss of final sigma expression contributes to malignant transformation by impairing the G(2) cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of final sigma expression are the most consistent molecular alterations in breast cancer identified so far.

Asunto(s)

Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Metilación de ADN , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Proteínas/genética , Tirosina 3-Monooxigenasa , Proteínas 14-3-3 , Azacitidina/análogos & derivados , Azacitidina/farmacología , Mama/citología , Neoplasias de la Mama/patología , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/fisiología , Línea Celular/efectos de los fármacos , Línea Celular Transformada/efectos de los fármacos , Transformación Celular Neoplásica/genética , Islas de CpG , Metilación de ADN/efectos de los fármacos , Reparación del ADN/genética , Decitabina , Células Epiteliales/metabolismo , Femenino , Fase G2/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Biosíntesis de Proteínas , Proteínas/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Neoplásico/biosíntesis , ARN Neoplásico/genética , Tolerancia a Radiación/genética , Proteínas Recombinantes de Fusión/fisiología , Transcripción Genética , Transfección , Células Tumorales Cultivadas/metabolismo