RESUMEN

The circadian system in mammals ensures adaptation to the light-dark cycle on Earth and imposes 24-h rhythmicity on metabolic, physiological and behavioral processes. The central circadian pacemaker is located in the brain and is entrained by environmental signals called Zeitgebers. From here, neural, humoral and systemic signals drive rhythms in peripheral clocks in nearly every mammalian tissue. During pregnancy, disruption of the complex interplay between the mother's rhythmic signals and the fetal developing circadian system can lead to long-term health consequences in the offspring. When an infant is born very preterm, it loses the temporal signals received from the mother prematurely and becomes totally dependent on 24/7 care in the Neonatal Intensive Care Unit (NICU), where day/night rhythmicity is usually blurred. In this literature review, we provide an overview of the fetal and neonatal development of the circadian system, and short-term consequences of disruption of this process as occurs in the NICU environment. Moreover, we provide a theoretical and molecular framework of how this disruption could lead to later-life disease. Finally, we discuss studies that aim to improve health outcomes after preterm birth by studying the effects of enhancing rhythmicity in light and noise exposure.

RESUMEN

The placenta is important in providing a healthy environment for the fetus and plays a central role in the pathophysiology of preeclampsia (PE). Fetal and placental developments are influenced by epigenetic programming. There is some evidence that PE is controlled to an altered circadian homeostasis. In a nested case-control study embedded in the Rotterdam Periconceptional Cohort, we obtained placental tissue, umbilical cord leukocytes (UCL), and human umbilical venous endothelial cells of 13 early-onset PE, 16 late-onset PE and 83 controls comprising 36 uncomplicated and 47 complicated pregnancies, i.e. 27 fetal growth restricted and 20 spontaneous preterm birth. To investigate the associations between PE and the epigenetics of circadian clock and clock-controlled genes in placental and newborn tissues, genome-wide DNA methylation analysis was performed using the Illumina HumanMethylation450K BeadChip and a candidate-gene approach using ANCOVA was applied on 939 CpGs of 39 circadian clock and clock-controlled genes. DNA methylation significantly differed in early-onset PE compared with spontaneous preterm birth at 6 CpGs in placental tissue (3.73E-5 ≤ p ≤ 0.016) and at 21 CpGs in UCL (1.09E-5≤ p ≤ 0.024). In early-onset PE compared with fetal growth restriction 2 CpGs in placental tissue (p < 0.05) and 8 CpGs in uncomplicated controls (4.78E-5≤ p ≤ 0.049) were significantly different. Moreover, significantly different DNA methylation in early-onset PE compared with uncomplicated controls was shown at 6 CpGs in placental tissue (1.36E-4≤ p ≤ 0.045) and 11 CpGs in uncomplicated controls (2.52E-6≤ p ≤ 0.009). No significant associations were shown with late-onset PE between study groups or tissues. The most differentially methylated CpGs showed hypomethylation in placental tissue and hypermethylation in uncomplicated controls. In conclusion, DNA methylation of circadian clock and clock-controlled genes demonstrated most differences in UCL of early-onset PE compared with spontaneous preterm birth. Implications of the tissue-specific variations in epigenetic programming for circadian performance and long-term health need further investigation.

Asunto(s)

Relojes Circadianos/genética , Péptidos y Proteínas de Señalización del Ritmo Circadiano/genética , Ritmo Circadiano/genética , Metilación de ADN , Epigénesis Genética , Placenta/metabolismo , Preeclampsia/genética , Adulto , Edad de Inicio , Estudios de Casos y Controles , Células Cultivadas , Péptidos y Proteínas de Señalización del Ritmo Circadiano/sangre , Islas de CpG , Femenino , Sangre Fetal/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Genotipo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Recién Nacido , Países Bajos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Preeclampsia/sangre , Preeclampsia/diagnóstico , Embarazo , Adulto Joven

RESUMEN

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants, and many are potent carcinogens. Benzo[a]pyrene (B[a]P), one of the best-studied PAHs, is metabolized ultimately to the genotoxin anti-B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE). BPDE triggers stress responses linked to gene expression, cell death and survival. So far, the underlying mechanisms that initiate these signal transduction cascades are unknown. Here we show that BPDE-induced DNA damage is recognized by DNA damage sensor proteins to induce activation of the stress-activated protein kinase (SAPK) p38. Surprisingly, the classical DNA damage response, which involves the kinases ATM and ATR, is not involved in p38-SAPK activation by BPDE. Moreover, the induction of p38-SAPK phosphorylation also occurs in the absence of DNA strand breaks. Instead, increased phosphorylation of p38-SAPK requires the nucleotide excision repair (NER) and DNA damage sensor proteins XPC and mHR23B. Interestingly, other genotoxins such as cisplatin (CDDP), hydrogen peroxide and ultraviolet radiation also enhance XPC-dependent p38-SAPK phosphorylation. In contrast, anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide, the DNA adducts of which are not properly recognized by NER, does not trigger p38-SAPK activation. As a downstream consequence, expression and secretion of the pro-inflammatory cytokine interleukin-6 is induced by BPDE and CDDP in vitro and by CDDP in the murine lung, and depends on XPC. In conclusion, we describe a novel pathway in which DNA damage recognition by NER proteins specifically leads to activation of p38-SAPK to promote inflammatory gene expression.

Asunto(s)

Carcinogénesis/metabolismo , Aductos de ADN/metabolismo , Reparación del ADN/fisiología , Interleucina-6/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/metabolismo , 7,8-Dihidro-7,8-dihidroxibenzo(a)pireno 9,10-óxido/toxicidad , Animales , Western Blotting , Carcinógenos/toxicidad , Ensayo Cometa , Daño del ADN/efectos de los fármacos , Daño del ADN/fisiología , Proteínas de Unión al ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática , Fibroblastos , Células HeLa , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutágenos/toxicidad , Células 3T3 NIH , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/fisiología , Transfección

RESUMEN

The genomic integrity of all living organisms is constantly jeopardized by physical [e.g. ultraviolet (UV) light, ionizing radiation] and chemical (e.g. environmental pollutants, endogenously produced reactive metabolites) agents that damage the DNA. To overcome the deleterious effects of DNA lesions, nature evolved a number of complex multi-protein repair processes with broad, partially overlapping substrate specificity. In marked contrast, cells may use very simple repair systems, referred to as direct DNA damage reversal, that rely on a single protein, remove lesions in a basically error-free manner, show high substrate specificity, and do not involve incision of the sugar-phosphate backbone or base excision. This concise review deals with two types of direct DNA damage reversal: (i) the repair of alkylating damage by alkyltransferases and dioxygenases, and (ii) the repair of UV-induced damage by spore photoproduct lyases and photolyases. (Part of a Multi-author Review).

Asunto(s)

Daño del ADN , Reparación del ADN , Modelos Moleculares , Transferasas Alquil y Aril/química , Transferasas Alquil y Aril/genética , Transferasas Alquil y Aril/metabolismo , Alquilantes/toxicidad , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/metabolismo , Dioxigenasas/química , Dioxigenasas/genética , Dioxigenasas/metabolismo , Filogenia , Rayos Ultravioleta/efectos adversos

RESUMEN

Members of the photolyase/cryptochrome family are flavoproteins that share an extraordinary conserved core structure (photolyase homology region, PHR), but the presence of a carboxy-terminal extension is limited to the cryptochromes. Photolyases are DNA-repair enzymes that remove UV-light-induced lesions. Cryptochromes of plants and Drosophila act as circadian photoreceptors, involved in light entrainment of the biological clock. Using knockout mouse models, mammalian cryptochromes (mCRY1 and mCRY2) were identified as essential components of the clock machinery. Within the mammalian transcription-translation feedback loop generating rhythmic gene expression, mCRYs potently inhibit the transcription activity of the CLOCK/BMAL1 heterodimer and protect mPER2 from 26S-protesome-mediated degradation. By analyzing a set of mutant mCRY1 proteins and photolyase/mCRY1 chimeric proteins, we found that the carboxyl terminus has a determinant role in mCRY1 function by harboring distinguished domains involved in nuclear import and interactions with other clock proteins. Moreover, the carboxyl terminus must cross-talk with the PHR to establish full transcription repression capacity in mCRY1. We propose that the presence of the carboxyl terminus in cryptochromes, which varies in sequence composition among mammalian, Drosophila, and plant CRYs, is critical for their different functions and possibly contributed to shape the different architecture and biochemistry of the clock machineries in these organisms.

Asunto(s)

Flavoproteínas/química , Flavoproteínas/fisiología , Animales , Ritmo Circadiano/genética , Ritmo Circadiano/fisiología , Criptocromos , Reparación del ADN , Desoxirribodipirimidina Fotoliasa/química , Desoxirribodipirimidina Fotoliasa/genética , Desoxirribodipirimidina Fotoliasa/fisiología , Dimerización , Flavoproteínas/genética , Fototransducción , Ratones , Ratones Noqueados , Modelos Biológicos , Fenotipo , Transcripción Genética

RESUMEN

In this study, we used an epidemiological approach to analyze an animal database of DNA repair deficient mice on reproductive performance in five Nucleotide Excision Repair (NER) mutant mouse models on a C57BL/6 genetic background, namely CSA, CSB, XPA, XPC [models for the human DNA repair disorders Cockayne Syndrome (CS) and xeroderma pigmentosum (XP), respectively] and mHR23B (not associated with human disease). This approach allowed us to detect and quantify reproductive effects based on a relatively small number of matings. We measured and quantified the scale of the effect between factors that might influence reproductive performance (i.e. age at co-housing, seasons) and reproductive parameters (i.e. litter size and pairing-to-birth interval -'pbi'). Besides, we detected and quantified the differences in reproductive performance between wild type mice and heterozygous/homozygous NER mutant mice. From our analyses, we found impaired reproduction in heterozygous and homozygous knock out mice; in particular, reduced litter size and lengthened pbi was related to the NER mutation-mHR23B, in heterozygous couples, even if they were otherwise phenotypically normal. Heterozygous mHR23B couples produced a 6.6-fold lower number of mHR23B(-/-) pups than indicated by Mendelian expectation; other genetic deficiencies studied were not statistically significant from each other or wild type controls. We concluded that careful epidemiological evaluations by analysis of animal database could provide reliable information on reproductive performance and detect deviations that would remain unnoticed without this. Also, some managerial aspects of mouse breeding could be evaluated.

Asunto(s)

ADN Helicasas/genética , Reparación del ADN/fisiología , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Proteínas/genética , Reproducción/fisiología , Proteína de la Xerodermia Pigmentosa del Grupo A/genética , Animales , Cruzamiento , Enzimas Reparadoras del ADN , Estudios Epidemiológicos , Femenino , Humanos , Tamaño de la Camada , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión a Poli-ADP-Ribosa , Estaciones del Año

RESUMEN

Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are rare autosomal recessive disorders associated with a defect in the nucleotide excision repair (NER) pathway required for the removal of DNA damage induced by UV light and distorting chemical adducts. Although progressive neurological dysfunction is one of the hallmarks of CS and of some groups of XP patients, the causative mechanisms are largely unknown. Here we show that mice lacking both the XPA (XP-group A) and CSB (CS-group B) genes in contrast to the single mutants display severe growth retardation, ataxia, and motor dysfunction during early postnatal development. Their cerebella are hypoplastic and showed impaired foliation and stunted Purkinje cell dendrites. Reduced neurogenesis and increased apoptotic cell death occur in the cerebellar external granular layer. These findings suggest that XPA and CSB have additive roles in the mouse nervous system and support a crucial role for these genes in normal brain development.

Asunto(s)

Ataxia/genética , Cerebelo/crecimiento & desarrollo , ADN Helicasas/fisiología , Reparación del ADN/genética , Proteínas de Unión al ADN/fisiología , Animales , Apoptosis , Conducta Animal , Cerebelo/patología , Síndrome de Cockayne/genética , ADN Helicasas/genética , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN/genética , Ratones , Ratones Noqueados , Proteínas de Unión a Poli-ADP-Ribosa , Xerodermia Pigmentosa/genética , Proteína de la Xerodermia Pigmentosa del Grupo A

RESUMEN

The stratum corneum and DNA repair do not completely protect keratinocytes from ultraviolet B. A third defense prevents cells with DNA photoproducts from becoming precancerous mutant cells: apoptosis of ultraviolet-damaged keratinocytes ("sunburn cells"). As signals for ultraviolet-induced apoptosis, some studies implicate DNA photoproducts in actively transcribed genes; other studies implicate non-nuclear signals. We traced and quantitated the in vivo DNA signal through several steps in the apoptosis-signaling pathway in haired mice. Homozygous inactivation of Xpa, Csb, or Xpc nucleotide excision repair genes directed the accumulation of DNA photoproducts to specific genome regions. Repair-defective Xpa-/- mice were 7-10-fold more sensitive to sunburn cell induction than wild-type mice, indicating that 86-90% of the ultraviolet B signal for keratinocyte apoptosis involved repairable photoproducts in DNA; the remainder involves unrepaired DNA lesions or nongenomic targets. Csb-/- mice, defective only in excising photoproducts from actively transcribed genes, were as sensitive as Xpa-/-, indicating that virtually all of the DNA signal originates from photoproducts in active genes. Conversely, Xpc-/- mice, defective in repairing the untranscribed majority of the genome, were as resistant to apoptosis as wild type. Sunburn cell formation requires the Trp53 tumor suppressor protein; 90-96% of the signal for its induction in vivo involved transcribed genes. Mdm2, which regulates the stability of Trp53 through degradation, was induced in vivo by low ultraviolet B doses but was suppressed at erythemal doses. DNA photoproducts in actively transcribed genes were involved in approximately 89% of the Mdm2 response.

Asunto(s)

Daño del ADN/fisiología , Proteínas Nucleares , Proteínas Proto-Oncogénicas/fisiología , Quemadura Solar/patología , Transcripción Genética/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Apoptosis/fisiología , ADN/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Eritema/etiología , Genoma , Ratones , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-mdm2 , Traumatismos por Radiación/complicaciones , Transducción de Señal/fisiología , Rayos Ultravioleta

RESUMEN

Ultraviolet B irradiation has serious consequences for cellular immunity and can suppress the rejection of skin tumors and the resistance to infectious diseases. DNA damage plays a crucial role in these immunomodulatory effects of ultraviolet B, as impaired repair of ultraviolet-B-induced DNA damage has been shown to cause suppression of cellular immunity. Ultraviolet-B-induced DNA damage is repaired by the nucleotide excision repair mechanism very efficiently. Nucleotide excision repair comprises two subpathways: transcription-coupled and global genome repair. In this study the immunologic consequences of specific nucleotide excision repair defects in three mouse models, XPA, XPC, and CSB mutant mice, were investigated. XPA mice carry a total nucleotide excision repair defect, whereas XPC and CSB mice only lack global genome and transcription-coupled nucleotide excision repair, respectively. Our data demonstrate that cellular immune parameters in XPA, XPC, and CSB mice are normal compared with their wild-type (control) littermates. This may indicate that the reported altered cellular responses in xeroderma pigmentosum patients are not constitutive but could be due to external factors, such as ultraviolet B. Upon exposure to ultraviolet B, only XPA mice are very sensitive to ultraviolet-B-induced inhibition of Th1-mediated contact hypersensitivity responses and interferon-gamma production in skin draining lymph nodes. Lipopolysaccharide-stimulated tumor necrosis factor alpha and interleukin-10 production are significantly augmented in both XPA and CSB mice after ultraviolet B exposure. Lymph node cell numbers were increased very significantly in XPA, mildly increased in CSB, and not in XPC mice. In general XPC mice do not exhibit any indication of enhanced ultraviolet B susceptibility with regard to the immune parameters analyzed. These data suggest that both global genome repair and transcription-coupled repair are needed to prevent immunomodulation by ultraviolet B, whereas transcription-coupled repair is the major DNA repair subpathway of nucleotide excision repair that prevents the acute ultraviolet-B-induced effects such as erythema.

Asunto(s)

Adyuvantes Inmunológicos/efectos de la radiación , ADN Helicasas/genética , Reparación del ADN/inmunología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ARN/genética , Proteínas Represoras/genética , Factores de Transcripción , Proteínas de Xenopus , Animales , Presentación de Antígeno/inmunología , Linfocitos B/inmunología , Reparación del ADN/genética , Enzimas Reparadoras del ADN , Hiperplasia , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión a Poli-ADP-Ribosa , Piel/inmunología , Piel/efectos de la radiación , Células TH1/inmunología , Células TH1/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Rayos Ultravioleta , Proteína de la Xerodermia Pigmentosa del Grupo A

RESUMEN

In mammals, the central circadian pacemaker resides in the hypothalamic suprachiasmatic nucleus (SCN), but circadian oscillators also exist in peripheral tissues. Here, using wild-type and cryptochrome (mCry)-deficient cell lines derived from mCry mutant mice, we show that the peripheral oscillator in cultured fibroblasts is identical to the oscillator in the SCN in (i) temporal expression profiles of all known clock genes, (ii) the phase of the various mRNA rhythms (i.e., antiphase oscillation of Bmal1 and mPer genes), (iii) the delay between maximum mRNA levels and appearance of nuclear mPER1 and mPER2 protein, (iv) the inability to produce oscillations in the absence of functional mCry genes, and (v) the control of period length by mCRY proteins.

Asunto(s)

Relojes Biológicos/genética , Ritmo Circadiano/genética , Proteínas de Unión al ADN , Proteínas de Drosophila , Proteínas del Ojo , Fibroblastos/fisiología , Regulación de la Expresión Génica , Células Fotorreceptoras de Invertebrados , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Proteínas CLOCK , Proteínas de Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Criptocromos , Endotelina-1/farmacología , Flavoproteínas/genética , Flavoproteínas/metabolismo , Perfilación de la Expresión Génica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptores Acoplados a Proteínas G , Núcleo Supraquiasmático/metabolismo , Factores de Tiempo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The hypothesis is advanced that the circadian pacemaker in the mammalian suprachiasmatic nucleus (SCN) is composed at the molecular level of a nonredundant double complex of circadian genes (per1, cry1, and per2, cry2). Each one of these sets would be sufficient for the maintenance of endogenous rhythmicity and thus constitute an oscillator. Each would have slightly different temporal dynamics and light responses. The per1/cry1 oscillator is accelerated by light and decelerated by darkness and thereby tracks dawn when day length changes. The per2 /cry2 oscillator is decelerated by light and accelerated by darkness and thereby tracks dusk. These M (morning) and E (evening) oscillators would give rise to the SCN's neuronal activity in an M and an E component. Suppression of behavioral activity by SCN activity in nocturnal mammals would give rise to adaptive tuning of the endogenous behavioral program to day length. The proposition-which is a specification of Pittendrigh and Daan's E-M oscillator model-yields specific nonintuitive predictions amenable to experimental testing in animals with mutations of circadian genes.

Asunto(s)

Ritmo Circadiano/genética , Estaciones del Año , Núcleo Supraquiasmático/fisiología , Animales , Animales Modificados Genéticamente , Electrofisiología , Luz , Ratones , Ratones Noqueados , Actividad Motora/fisiología

RESUMEN

Cells isolated from individuals with Cockayne syndrome (CS) have a defect in transcription-coupled DNA repair, which rapidly corrects certain DNA lesions located on the transcribed strand of active genes. Despite this DNA repair defect, individuals with CS group A (CSA) or group B (CSB) do not exhibit an increased spontaneous or UV-induced cancer rate. In order to investigate the effect of CSB deficiency on spontaneous carcinogenesis, we crossed CSB(-/-) mice with cancer-prone mice lacking the p16(Ink4a)/p19(ARF) tumor suppressor locus. CSB(-/-) mice are sensitive to UV-induced skin cancer but show no increased rate of spontaneous cancer. CSB(-/-) Ink4a/ARF(-/-) mice developed 60% fewer tumors than Ink4a/ARF(-/-) animals and demonstrated a longer tumor-free latency time (260 versus 150 days). Moreover, CSB(-/-) Ink4a/ARF(-/-) mouse embryo fibroblasts (MEFs) exhibited a lower colony formation rate after low-density seeding, a lower rate of H-Ras-induced transformation, slower proliferation, and a lower mRNA synthesis rate than Ink4a/ARF(-/-) MEFs. CSB(-/-) Ink4a/ARF(-/-) MEFs were also more sensitive to UV-induced p53 induction and UV-induced apoptosis than were Ink4a/ARF(-/-) MEFs. In order to investigate whether the apparent antineoplastic effect of CSB gene disruption was caused by sensitization to genotoxin-induced (p53-mediated) apoptosis or by p53-independent sequelae, we also generated p53(-/-) and CSB(-/-) p53(-/-) MEFs. The CSB(-/-) p53(-/-) MEFs demonstrated lower colony formation efficiency, a lower proliferation rate, a lower mRNA synthesis rate, and a higher rate of UV-induced cell death than p53(-/-) MEFs. Collectively, these results indicate that the antineoplastic effect of CSB gene disruption is at least partially p53 independent; it may result from impaired transcription or from apoptosis secondary to environmental or endogenous DNA damage.

Asunto(s)

Síndrome de Cockayne/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , ADN Helicasas/genética , ADN Helicasas/fisiología , Neoplasias/genética , Proteínas/genética , Factores de Edad , Animales , Apoptosis , División Celular , Cruzamientos Genéticos , Reparación del ADN , Enzimas Reparadoras del ADN , Fibroblastos/metabolismo , Fibrosarcoma/metabolismo , Citometría de Flujo , Genes p53/genética , Predisposición Genética a la Enfermedad , Genotipo , Immunoblotting , Etiquetado Corte-Fin in Situ , Linfoma/metabolismo , Ratones , Ratones Noqueados , Proteínas de Unión a Poli-ADP-Ribosa , ARN Mensajero/metabolismo , Factores de Tiempo , Transformación Genética , Proteína p14ARF Supresora de Tumor , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta , Proteínas ras/metabolismo

Asunto(s)

Relojes Biológicos/genética , Proteínas Nucleares/genética , Núcleo Supraquiasmático/metabolismo , Animales , Proteínas de Ciclo Celular , Ritmo Circadiano/genética , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes , Ratones , Proteínas Circadianas Period

RESUMEN

Cockayne syndrome (CS) patients are deficient in the transcription coupled repair (TCR) subpathway of nucleotide excision repair (NER) but in contrast to xeroderma pigmentosum patients, who have a defect in the global genome repair subpathway of NER, CS patients do not have an elevated cancer incidence. To determine to what extent a TCR deficiency affects carcinogen-induced mutagenesis and carcinogenesis, CS group B correcting gene (CSB)-deficient mice were treated with the genotoxic carcinogen benzo(a)pyrene (B[a]P) at an oral dose of 13 mg/kg body weight, three times a week. At different time points, mutant frequencies at the inactive lacZ gene (in spleen, liver, and lung) as well as at the active hypoxanthine phosphoribosyltransferase (Hprt) gene (in spleen) were determined to compare mutagenesis at inactive versus active genes. B[a]P treatment gave rise to increased mutant frequencies at lacZ in all of the organs tested without a significant difference between CSB-/- and wild-type mice, whereas B[a]P-induced Hprt mutant frequencies in splenic T-lymphocytes were significantly more enhanced in CSB-/- mice than in control mice. The sequence data obtained from Hprt mutants indicate that B[a]P adducts at guanine residues were preferentially removed from the transcribed strand of the Hprt gene in control mice but not in CSB-/- mice. On oral treatment with B[a]P, the tumor incidence increased in both wild-type and CSB-deficient animals. However, no differences in tumor rate were observed between TCR-deficient CSB-/- mice and wild-type mice, which is in line with the normal cancer susceptibility of CS patients. The mutagenic response at lacZ, in contrast to Hprt, correlated well with the cancer incidence in CSB-/- mice after B[a]P treatment, which suggests that mutations in the bulk of the DNA (inactive genes) are a better predictive marker for carcinogen-induced tumorigenesis than mutations in genes that are actively transcribed. Thus, the global genome repair pathway of NER appears to play an important role in the prevention of cancer.

Asunto(s)

Benzo(a)pireno/toxicidad , Carcinógenos/toxicidad , Cocarcinogénesis , Síndrome de Cockayne/genética , Reparación del ADN/genética , Mutagénesis/efectos de los fármacos , Neoplasias Experimentales/etiología , Animales , Cruzamientos Genéticos , ADN/genética , Femenino , Expresión Génica , Predisposición Genética a la Enfermedad/genética , Hipoxantina Fosforribosiltransferasa/genética , Operón Lac/efectos de los fármacos , Operón Lac/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutagénesis/genética , Neoplasias Experimentales/inducido químicamente , Neoplasias Experimentales/genética , Valor Predictivo de las Pruebas , Transcripción Genética/genética

RESUMEN

DNA damages caused by cellular metabolites and environmental agents induce mutations, that may predispose to cancer. Nucleotide excision repair (NER) is a major cellular defence mechanism acting on a variety of DNA lesions. Here, we show that spontaneous mutant frequencies at the Hprt gene increased 30-fold in T-lymphocytes of 1 year old Xpc-/- mice, possessing only functional transcription-coupled repair (TCR). Hprt mutant frequencies in Xpa-/- and Csb-/- mice that both have a defect in this NER subpathway, remained low during ageing. In contrast to current models, the elevated mutation rate in Xpc-/- mice does not lead to an increased tumour incidence or premature ageing. Oncogene (2000) 19, 5034 - 5037

Asunto(s)

Envejecimiento/genética , Reparación del ADN/genética , Mutagénesis , Xerodermia Pigmentosa/genética , Animales , Femenino , Predisposición Genética a la Enfermedad , Hipoxantina Fosforribosiltransferasa/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Neoplasias Experimentales/genética , Bazo/citología , Linfocitos T/fisiología , Transcripción Genética/genética

RESUMEN

Nucleotide excision repair (NER), apoptosis, and cell-cycle regulation are major defense mechanisms against the carcinogenic effects of UVB light. NER eliminates UVB-induced DNA photolesions via two subpathways: global genome repair (GGR) and transcription-coupled repair (TCR). Defects in NER result in the human disorders xeroderma pigmentosum (XP) and Cockayne syndrome (CS), displaying severe UV sensitivity and in the case of XP, cancer proneness. We investigated the impact of deficiencies in NER subpathways on apoptosis, hyperplasia, and cell cycle progression in the epidermis of UVB-exposed CS group B (Csb(-/-)) mice (no TCR), XP group C (Xpc(-/-)) mice (no GGR), and XP group A (Xpa(-/-)) mice (no TCR and no GGR). On UVB treatment (250 J/m(2)), Xpa(-/-) and Csb(-/-) mice revealed an extensive apoptotic response in the skin, a blockage of cell cycle progression of epidermal cells, and strong hyperplasia. Interestingly, the absence of this apoptotic response in the skin of wild-type and Xpc(-/-) mice coincided with the ability of epidermal cells to enter the S phase. However, only epidermal cells of Xpc(-/-) mice subsequently became arrested in the G(2) phase. Our data demonstrate that TCR (and/or restoration of UVB-inhibited transcription) enables damaged cells to progress through S phase and prevents the induction of apoptosis and hyperplasia. G(2) arrest is manifest only under conditions of proficient TCR in combination with deficient GGR, indicating that epidermal cells become arrested in the G(2) phase as a result of persisting damage in their genome.

Asunto(s)

Apoptosis/efectos de la radiación , Reparación del ADN/genética , Epidermis/efectos de la radiación , Fase G2/efectos de la radiación , Transcripción Genética , Animales , Células Epidérmicas , Humanos , Ratones , Ratones Pelados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/fisiología , Fase S , Rayos Ultravioleta

RESUMEN

Nuclear entry of circadian oscillatory gene products is a key step for the generation of a 24-hr cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. Previously, mCRY1 and mCRY2 have been found to complex with PER proteins leading to nuclear import. Here we report that nuclear translocation of mPER1 and mPER2 (1) involves physical interactions with mPER3, (2) is accelerated by serum treatment, and (3) still occurs in mCry1/mCry2 double-deficient cells lacking a functional biological clock. Moreover, nuclear localization of endogenous mPER1 was observed in cultured mCry1/mCry2 double-deficient cells as well as in the liver and the suprachiasmatic nuclei (SCN) of mCry1/mCry2 double-mutant mice. This indicates that nuclear translocation of at least mPER1 also can occur under physiological conditions (i.e., in the intact mouse) in the absence of any CRY protein. The mPER3 amino acid sequence predicts the presence of a cytoplasmic localization domain (CLD) and a nuclear localization signal (NLS). Deletion analysis suggests that the interplay of the CLD and NLS proposed to regulate nuclear entry of PER in Drosophila is conserved in mammals, but with the novel twist that mPER3 can act as the dimerizing partner.

Asunto(s)

Núcleo Celular/metabolismo , Proteínas de Drosophila , Proteínas del Ojo , Células Fotorreceptoras de Invertebrados , Animales , Células COS , Proteínas de Ciclo Celular , Línea Celular , Criptocromos , Medio de Cultivo Libre de Suero , Citoplasma/metabolismo , Dimerización , Fibroblastos/metabolismo , Flavoproteínas/metabolismo , Immunoblotting , Inmunohistoquímica , Hígado/metabolismo , Ratones , Ratones Noqueados , Mutagénesis , Señales de Localización Nuclear , Proteínas Nucleares/metabolismo , Proteínas Circadianas Period , Pruebas de Precipitina , Ratas , Receptores Acoplados a Proteínas G , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción

RESUMEN

The nucleotide excision repair (NER) system is comprised of two subpathways, i.e., transcription-coupled repair (TCR) and global genome repair (GGR). To establish the relative importance of TCR and GGR for UV effects on the skin, we have used hairless knockout mouse strain lacking either TCR (CSB -/-) or GGR (XPC -/-). In single exposure experiments, we found that CSB -/- mice have a 7-16 times higher susceptibility to sunburn than XPC -/- mice and than heterozygous (+/-) and wild-type (+/+) controls. Exposure to 80 J/m2 UV radiation (i.e., suberythemogenic in CSB -/-) on 10 consecutive days gives rise to epidermal hyperplasia in CSB -/- and XPC -/-, whereas repair-proficient controls do not show epidermal hyperplasia from these exposures. In addition, CSB -/- mice develop marked parakeratosis, whereas XPC -/- mice and controls do not. Under continued exposure to this daily dose, squamous cell carcinomas appear in CSB -/-, XPC -/-, and in the control groups, whereas only in the CSB -/- animals is a fairly high number of benign papillomas also found. The median latency time of squamous cell carcinomas (diameters > or = 1 mm) is 84 days for the XPC -/- mice, 115 days for the CSB -/- mice, and 234-238 days for the heterozygous and wild-type control groups. These results indicate that GGR is more important than TCR in protection against UV-induced carcinomas of the skin but not against other UV effects such as sunburn, epidermal thickening, scaling of the stratum corneum, and development of papillomas. These results also indicate that GGR capacity may serve as a better predictor for skin cancer susceptibility than sensitivity to sunburn. The relative cancer susceptibilities of GGR- and TCR-deficient skin could well depend on the balance between an increased mutation rate and the presence (in CSB -/-) or lack (in XPC -/-) of a compensatory apoptotic response.

Asunto(s)

Reparación del ADN , Transcripción Genética , Rayos Ultravioleta , Animales , Apoptosis , Carcinoma de Células Escamosas/etiología , Carcinoma de Células Escamosas/genética , Epidermis/patología , Epidermis/efectos de la radiación , Exones , Ratones , Ratones Pelados , Ratones Noqueados , Mutación , Neoplasias Inducidas por Radiación/etiología , Neoplasias Inducidas por Radiación/genética , Papiloma/etiología , Papiloma/genética , Paraqueratosis/etiología , Paraqueratosis/genética , Neoplasias Cutáneas/etiología , Neoplasias Cutáneas/genética , Quemadura Solar/genética , Factores de Tiempo

RESUMEN

Exposure to UV-B radiation impairs immune responses in mammals by inhibiting especially Th1-mediated contact hypersensitivity and delayed-type hypersensitivity. Immunomodulation is not restricted to the exposed skin, but is also observed at distant sites, indicating the existence of mediating factors such as products from exposed skin cells or photoactivated factors present in the superficial layers. DNA damage appears to play a key role, because enhanced nucleotide excision repair (NER) strongly counteracts immunosuppression. To determine the effects of the type and genomic location of UV-induced DNA damage on immunosuppression and acute skin reactions (edema and erythema) four congenic mouse strains carrying different defects in NER were compared: CSB and XPC mice lacking transcription-coupled or global genome NER, respectively, as well as XPA and TTD/XPD mice carrying complete or partial defects in both NER subpathways, respectively. The major conclusions are that 1) transcription-coupled DNA repair is the dominant determinant in protection against acute skin effects; 2) systemic immunomodulation is only affected when both NER subpathways are compromised; and 3) sunburn is not related to UV-B-induced immunosuppression.

Asunto(s)

Reparación del ADN/inmunología , Genoma , Terapia de Inmunosupresión , Piel/inmunología , Piel/efectos de la radiación , Transcripción Genética/inmunología , Rayos Ultravioleta , Animales , Síndrome de Cockayne/genética , Síndrome de Cockayne/inmunología , Reparación del ADN/efectos de la radiación , Dermatitis por Contacto/genética , Dermatitis por Contacto/inmunología , Relación Dosis-Respuesta Inmunológica , Relación Dosis-Respuesta en la Radiación , Edema/genética , Edema/inmunología , Eritema/genética , Eritema/inmunología , Enfermedades del Cabello/genética , Enfermedades del Cabello/inmunología , Hipersensibilidad Tardía/genética , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Tardía/microbiología , Listeria monocytogenes/inmunología , Listeria monocytogenes/efectos de la radiación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Cloruro de Picrilo/inmunología , Piel/metabolismo , Transcripción Genética/efectos de la radiación , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/inmunología

RESUMEN

We show that, in the mouse, the core mechanism for the master circadian clock consists of interacting positive and negative transcription and translation feedback loops. Analysis of Clock/Clock mutant mice, homozygous Period2(Brdm1) mutants, and Cryptochrome-deficient mice reveals substantially altered Bmal1 rhythms, consistent with a dominant role of PERIOD2 in the positive regulation of the Bmal1 loop. In vitro analysis of CRYPTOCHROME inhibition of CLOCK: BMAL1-mediated transcription shows that the inhibition is through direct protein:protein interactions, independent of the PERIOD and TIMELESS proteins. PERIOD2 is a positive regulator of the Bmal1 loop, and CRYPTOCHROMES are the negative regulators of the Period and Cryptochrome cycles.

Asunto(s)

Relojes Biológicos/fisiología , Ritmo Circadiano/fisiología , Proteínas de Drosophila , Proteínas del Ojo , Flavoproteínas/metabolismo , Proteínas Nucleares/metabolismo , Células Fotorreceptoras de Invertebrados , Núcleo Supraquiasmático/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción ARNTL , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Relojes Biológicos/genética , Proteínas CLOCK , Proteínas de Ciclo Celular , Línea Celular , Núcleo Celular/metabolismo , Ritmo Circadiano/genética , Criptocromos , Dimerización , Retroalimentación , Femenino , Flavoproteínas/genética , Regulación de la Expresión Génica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos Biológicos , Mutación , Proteínas Nucleares/genética , Proteínas Circadianas Period , Biosíntesis de Proteínas , ARN/metabolismo , Receptores Acoplados a Proteínas G , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Transcripción Genética