RESUMO
In central Brazil, in the municipality of Faina (state of Goiás), the small and isolated village of Araras comprises a genetic cluster of xeroderma pigmentosum (XP) patients. The high level of consanguinity and the geographical isolation gave rise to a high frequency of XP patients. Recently, two founder events were identified affecting that community, with two independent mutations at the POLH gene, c.764 + 1 G > A (intron 6) and c.907 C > T; p.Arg303* (exon 8). These deleterious mutations lead to the xeroderma pigmentosum variant syndrome (XP-V). Previous reports identified both mutations in other countries: the intron 6 mutation in six patients (four families) from Northern Spain (Basque Country and Cantabria) and the exon 8 mutation in two patients from different families in Europe, one of them from Kosovo. In order to investigate the ancestry of the XP patients and the age for these mutations at Araras, we generated genotyping information for 22 XP-V patients from Brazil (16), Spain (6) and Kosovo (1). The local genomic ancestry and the shared haplotype segments among the patients showed that the intron 6 mutation at Araras is associated with an Iberian genetic legacy. All patients from Goiás, homozygotes for intron 6 mutation, share with the Spanish patients identical-by-descent (IBD) genomic segments comprising the mutation. The entrance date for the Iberian haplotype at the village was calculated to be approximately 200 years old. This result is in agreement with the historical arrival of Iberian individuals at the Goiás state (BR). Patients from Goiás and the three families from Spain share 1.8 cM (family 14), 1.7 cM (family 15), and a more significant segment of 4.7 cM within family 13. On the other hand, the patients carrying the exon 8 mutation do not share any specific genetic segment, indicating an old genetic distance between them or even no common ancestry.
Assuntos
DNA Polimerase Dirigida por DNA/genética , Haplótipos , Padrões de Herança , Mutação , Isolamento Reprodutivo , Xeroderma Pigmentoso/genética , Brasil/epidemiologia , Consanguinidade , Europa (Continente)/epidemiologia , Éxons , Feminino , Genética Populacional , Heterozigoto , Homozigoto , Migração Humana , Humanos , Íntrons , Masculino , Fenótipo , Xeroderma Pigmentoso/epidemiologia , Xeroderma Pigmentoso/patologiaAssuntos
Proteínas de Ligação a DNA/genética , Efeito Fundador , Neoplasias Cutâneas/genética , Proteína Grupo D do Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/genética , Adolescente , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Mutação , Neoplasias Cutâneas/patologia , África do Sul , Xeroderma Pigmentoso/patologia , Adulto JovemRESUMO
BACKGROUND: Xeroderma pigmentosum (XP) is a rare human syndrome associated with hypersensitivity to sunlight and a high frequency of skin tumours at an early age. We identified a community in the state of Goias (central Brazil), a sunny and tropical region, with a high incidence of XP (17 patients among approximately 1000 inhabitants). OBJECTIVES: To identify gene mutations in the affected community and map the distribution of the affected alleles, correlating the mutations with clinical phenotypes. METHODS: Functional analyses of DNA repair capacity and cell-cycle responses after ultraviolet exposure were investigated in cells from local patients with XP, allowing the identification of the mutated gene, which was then sequenced to locate the mutations. A specific assay was designed for mapping the distribution of these mutations in the community. RESULTS: Skin primary fibroblasts showed normal DNA damage removal but abnormal DNA synthesis after ultraviolet irradiation and deficient expression of the Polη protein, which is encoded by POLH. We detected two different POLH mutations: one at the splice donor site of intron 6 (c.764 +1 G>A), and the other in exon 8 (c.907 C>T, p.Arg303X). The mutation at intron 6 is novel, whereas the mutation at exon 8 has been previously described in Europe. Thus, these mutations were likely brought to the community long ago, suggesting two founder effects for this rare disease. CONCLUSIONS: This work describes a genetic cluster involving POLH, and, particularly unexpected, with two independent founder mutations, including one that likely originated in Europe.
Assuntos
Efeito Fundador , Mutação/genética , Neoplasias Cutâneas/genética , Xeroderma Pigmentoso/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Brasil/etnologia , Europa (Continente)/etnologia , Feminino , Heterozigoto , Homozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Células Tumorais Cultivadas , Xeroderma Pigmentoso/etnologiaRESUMO
BACKGROUND: Xeroderma pigmentosum type C (XP-C) is a rare, autosomal, recessive condition characterized by the association of various clinical manifestations mostly involving the skin and eyes. OBJECTIVES: To evaluate the clinical manifestations in a homogeneous, genetically characterized cohort of patients with XP-C. METHODS: All patients with XP-C, which was confirmed genetically or by unscheduled DNA synthesis, from the registry of our department and from the French association of patients 'Les Enfants de la Lune' were contacted. During a planned consultation, clinical information was collected using a standardized case-record form. RESULTS: In total, 31 patients were seen. The mean age at diagnosis was 2.95 years; skin symptoms started at a mean age of 1.49 years. Among the patients, 52% had relatively short stature, with a height-for-weight z-score below -1 SD; 62% showed pyramidal syndrome and 45% had photophobia and/or conjunctivitis. Four patients had several pyogenic granulomas. Twenty-four patients (77%) had skin cancer. The mean age of onset of the first skin cancer was 4.76 years (range 2-14.5 years). Basal-cell carcinoma was the most frequent cancer. Melanomas were rare and mostly desmoplastic. Multinodular thyroid was the most frequent internal tumour. CONCLUSIONS: Our data highlight several new aspects of XP-C. Patients with XP-C are at risk of developing pyogenic granulomas, desmoplastic melanomas and multinodular thyroid. Involvement of the central nervous system is frequent, but its mechanism remains unclear. The relatively short stature of the patients needs further investigation in order to be explained. XP-C is not only a cancer-prone disorder but is also a polysystemic disorder.
Assuntos
Carcinoma Basocelular/diagnóstico , Granuloma Piogênico/diagnóstico , Melanoma/diagnóstico , Neoplasias Cutâneas/diagnóstico , Neoplasias da Glândula Tireoide/diagnóstico , Xeroderma Pigmentoso/diagnóstico , Adolescente , Carcinoma Basocelular/genética , Criança , Pré-Escolar , Estudos de Coortes , Feminino , França , Granuloma Piogênico/genética , Humanos , Masculino , Melanoma/genética , Neoplasias Cutâneas/genética , Neoplasias da Glândula Tireoide/genética , Xeroderma Pigmentoso/genéticaRESUMO
We have designed an amphiphilic prodrug of gemcitabine (dFdC) by its covalent coupling to a derivative of squalene, a natural lipid. The resulting bioconjugate self-assembled spontaneously in water as nanoparticles that displayed a promising in vivo anticancer activity. The aim of the present study was to provide further insight into the in vitro subcellular localization and on the metabolization pathway of the prodrug. Cells treated with radiolabelled squalenoyl gemcitabine (SQdFdC) were studied by differential detergent permeation, and microautography coupled to fluorescent immunolabeling and confocal microscopy. This revealed that the bioconjugate accumulated within cellular membranes, especially in those of the endoplasmic reticulum. Radio-chromatography analysis proved that SQdFdC delivered dFdC directly in the cell cytoplasm. Mass spectrometry studies confirmed that gemcitabine was then either converted into its biologically active triphosphate metabolite or exported from the cells through membrane transporters. To our knowledge, this is the first description of such an intracellular drug delivery pathway. In vitro cytotoxicity assays revealed that SQdFdC was more active than dFdC on a transporter-deficient human resistant leukemia model, which was explained by the subcellular distribution of the drugs and their metabolites. The squalenoylation drug delivery strategy might, therefore, dramatically improve the efficacy of gemcitabine on transporter-deficient resistant cancer in the clinical context.
Assuntos
Antimetabólitos Antineoplásicos/farmacocinética , Membrana Celular/metabolismo , Desoxicitidina/análogos & derivados , Portadores de Fármacos/química , Nanopartículas/química , Pró-Fármacos/farmacocinética , Esqualeno/análogos & derivados , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Autorradiografia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacocinética , Desoxicitidina/farmacologia , Composição de Medicamentos , Humanos , Tamanho da Partícula , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacologia , Esqualeno/administração & dosagem , Esqualeno/farmacocinética , Esqualeno/farmacologia , Frações Subcelulares/metabolismo , Tensoativos/química , Espectrometria de Massas em Tandem , Distribuição Tecidual , GencitabinaRESUMO
Melanoma causes a considerable public health burden because of its dramatic rise in incidence worldwide since the mid-1960s and because the metastatic disease remains incurable, has a short median survival and is characterized by resistance to almost all classes of cytotoxic agents. DNA repair pathways are multiple and are able to repair, usually in an error-free manner, all kinds of DNA damage induced by exogenous and endogenous genotoxic agents. This review describes the role of DNA repair process in protecting us from cancer and particularly nucleotide excision deficiencies that are associated with melanoma development. Resistance of tumoral cells to antitumoral regimen can be caused by overexpression of DNA repair processes. We showed that melanoma metastasis was associated with higher expression of some DNA repair pathways leading to a better surveillance of replication fork fidelity. We showed a partially coordinated regulation of these repair genes. P53 and several transcription factors may regulate numerous of these repair genes. The repair pathways that are overexpressed in metastatic melanoma are those particularly efficient in repairing the major DNA damage produced by cytotoxic treatments. This implies that better analysis of DNA repair regulation is necessary to identify novel therapeutic targets and to allow clinicians to propose tailored therapies.
Assuntos
Reparo do DNA , Melanoma/secundário , Dano ao DNA/genética , Reparo do DNA/genética , Reparo do DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Melanoma/tratamento farmacológico , Melanoma/etiologia , Melanoma/genética , Modelos Genéticos , Polimorfismo de Nucleotídeo Único , Fatores de Risco , Luz Solar/efeitos adversosRESUMO
DNA repair mechanisms constitute major defences against agents that cause cancer, degenerative disease and aging. Different repair systems cooperate to maintain the integrity of genetic information. Investigations of DNA repair involvement in human pathology require an efficient tool that takes into account the variety and complexity of repair systems. We have developed a highly sensitive damaged plasmid microarray to quantify cell lysate excision/synthesis (ES) capacities using small amounts of proteins. This microsystem is based on efficient immobilization and conservation on hydrogel coated glass slides of plasmid DNA damaged with a panel of genotoxic agents. Fluorescent signals are generated from incorporation of labelled dNTPs by DNA excision-repair synthesis mechanisms at plasmid sites. Highly precise DNA repair phenotypes i.e. simultaneous quantitative measures of ES capacities toward seven lesions repaired by distinct repair pathways, are obtained. Applied to the characterization of xeroderma pigmentosum (XP) cells at basal level and in response to a low dose of UVB irradiation, the assay showed the multifunctional role of different XP proteins in cell protection against all types of damage. On the other hand, measurement of the ES of peripheral blood mononuclear cells from six donors revealed significant diversity between individuals. Our results illustrate the power of such a parallelized approach with high potential for several applications including the discovery of new cancer biomarkers and the screening of chemical agents modulating DNA repair systems.
Assuntos
Reparo do DNA , Plasmídeos , Linhagem Celular Transformada , Células HeLa , Humanos , Espectrometria de FluorescênciaRESUMO
We report the case of an 83-year-old French woman with multiple melanomas showing a severe DNA repair deficiency, corrected after transfection by XPC cDNA. Two biallelic mutations in the XPC gene are reported: an inactivating frameshift mutation in exon 15 (c.2544delG, p.W848X) and a missense mutation in exon 11 (c.2108 C>T, P703L). We demonstrate that these new mutations are involved in the DNA repair deficiency and confirm the diagnosis of xeroderma pigmentosum from complementation group C (XP-C). We speculate that the coexistence of a MC1R variant may be involved in the phenotype of multiple melanomas and that the unusual long-term survival may be related to a lower ultraviolet radiation exposure and to a regular clinical follow-up. This patient appears to be the first French Caucasian XP-C case and one of the oldest living patients with XP reported worldwide.
Assuntos
Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Mutação da Fase de Leitura/genética , Melanoma/genética , Mutação de Sentido Incorreto/genética , Neoplasias Primárias Múltiplas/genética , Neoplasias Cutâneas/genética , Xeroderma Pigmentoso/genética , Idoso de 80 Anos ou mais , Feminino , Humanos , Melanoma/patologia , Neoplasias Primárias Múltiplas/patologia , Fenótipo , Neoplasias Cutâneas/patologia , Sobreviventes , População Branca , Xeroderma Pigmentoso/patologiaRESUMO
Xeroderma pigmentosum (XP) is a rare, recessively inherited genetic disease characterized by skin cancer proneness and premature aging in photoexposed area. The disease results from defective nucleotide excision repair of ultraviolet (UV)-induced DNA lesions. Reconstruction of group C (XP-C) skin in vitro previously suggested that patients' dermal fibroblasts might be involved in promoting skin cancer development, as they elicited microinvasions of both control and XP-C keratinocytes within dermal equivalents. Here we show that in the absence of UV exposure XP-C fibroblasts exhibit aged-like features such as an elongated and dendritic shape. We analysed the repertoire of expression of matrix metalloproteinases (MMPs) involved in skin aging and cancer. All XP-C fibroblasts tested in this study overexpressed specifically and significantly MMP1. MMP1 expression was also found increased in the dermis of XP-C skin sections suggesting the active contribution of XP-C mesenchymal cells to skin aging and exacerbated carcinogenesis. Increased MMP1 expression in cultured XP-C fibroblasts resulted from MMP1 mRNA accumulation and enhanced transcriptional activity of the MMP1 gene promoter. Deletion analysis revealed the essential role of AP-1 activation in constitutive MMP1 overexpression in XP-C primary fibroblasts. In parallel, levels of reactive oxygen species and FOSB DNA-binding activity were found increased in XP-C fibroblasts. Altogether, these observations suggest that beyond its role in nucleotide excision repair the XPC protein may be important in cell metabolism and fate in the absence of UV.
Assuntos
Reparo do DNA , Metaloproteinase 1 da Matriz/metabolismo , Pele/enzimologia , Xeroderma Pigmentoso/enzimologia , Fibroblastos/enzimologia , Humanos , Metaloproteinase 1 da Matriz/genética , Regiões Promotoras Genéticas , Espécies Reativas de Oxigênio/metabolismo , Pele/patologia , Transcrição Gênica , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologiaRESUMO
We have identified a gene-profile signature for human primary malignant melanoma associated with metastasis to distant sites and poor prognosis. We analyse the differential gene expression by looking at whole biological pathways rather than individual genes. Among the most significant pathways associated with progression to metastasis, we found the DNA replication (P=10(-14)) and the DNA repair pathways (P=10(-16)). We concentrated our analysis on DNA repair and found that 48 genes of this category, among a list of 234 genes, are associated with metastatic progression. These genes belong essentially to the pathways allowing recovery of stalled replication forks due to spontaneous blockage or induced DNA lesions. Because almost all these differentially expressed repair genes were overexpressed in primary tumors with bad prognosis, we speculate that primary melanoma cells that will metastasize try to replicate in a fast and error-free mode. In contrast to the progression from melanocytes to primary melanoma, genetic stability appears to be necessary for a melanoma cell to give rise to distant metastasis. This overexpression of repair genes explains nicely the extraordinary resistance of metastatic melanoma to chemo- and radio-therapy. Our results may open a new avenue for the discovery of drugs active on human metastatic melanoma.
Assuntos
Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Transformação Celular Neoplásica , Perfilação da Expressão Gênica , HumanosRESUMO
Accumulation of frameshift mutations at genes containing coding mononucleotide repeats is thought to be the major molecular mechanism by which mismatch repair-deficient cells accumulate functional alterations. These mutations resulting from microsatellite instability (MSI) can affect genes involved in pathways with a putative oncogenic role, but may also arise in genes without any expected role in MSI carcinogenesis because of the high mutation background of these tumours. We here screened 39 MSI colorectal tumours for the presence of mutations in 25 genes involved in DNA damage signalling and repair pathways. Using a maximum likelihood statistical method, these genes were divided into two different groups that differed significantly in their mutation frequencies, and likely represent mutations that do or do not provide selective pressure during MSI tumour progression. Interestingly, the so-called real-target mutational events were found to be distributed among genes involved in different functional pathways of the DNA metabolism, for example, DNA damage signalling (DNA-PKcs, ATR), double-strand break (DSB) repair (DNA-PKcs, RAD50), mismatch repair (MSH3, MSH6, MBD4) and replication (POLD3). In particular, mutations in MRE11 and/or RAD50 were observed in the vast majority of the tumours and resulted in the concomitant loss of immunohistochemical expression of both proteins. These data might explain why MSI colorectal cancers (CRC) behave differently in response to a wide variety of chemotherapeutic agents, notably those targeting DNA. More generally, they give further insights into how MSI leads to functional changes with synergistic effects in oncogenic pathways.
Assuntos
Neoplasias Colorretais/genética , Dano ao DNA , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Instabilidade de Microssatélites , Transdução de Sinais , Adulto , Idoso , Antineoplásicos/farmacologia , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
To characterize further the role of the INK4a-ARF locus in the multistep process of skin carcinogenesis, we performed a mutational analysis of this locus in skin lesions from hairless mice either irradiated with UVB alone or with a solar simulator delivering UVA + B. INK4a-ARF mutations were present in five of 57 squamous cell carcinomas (9%), but no mutation was detected in precancerous lesions. All mutations were C:G > T:A transitions located at dipyrimidic sites, the hallmark of UVB mutagenesis. Three mutations affected only the p19(ARF) reading frame, whereas two mutations affected only the p16(INK4a) transcript. This study demonstrates for the first time UV-induced mutations of INK4a-ARF that occur in a small percentage in late stages skin tumors.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Mutação , Neoplasias Induzidas por Radiação/genética , Neoplasias Cutâneas/genética , Proteína Supressora de Tumor p14ARF/genética , Raios Ultravioleta/efeitos adversos , Animais , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/genética , Dano ao DNA/efeitos da radiação , Genes p16 , Camundongos , Camundongos Pelados , Neoplasias Induzidas por Radiação/etiologia , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Lesões Pré-Cancerosas/etiologia , Lesões Pré-Cancerosas/genética , Pirimidinas/metabolismo , Neoplasias Cutâneas/etiologia , Proteína Supressora de Tumor p53/genéticaRESUMO
INTRODUCTION: Xeroderma pigmentosum is a relatively frequent genodermatosis in North Africa. It is characterized by abnormal sensitivity to ultraviolet light, responsible for the early occurrence of multiple cutaneous neoplasms. We present the results of the clinical and biological investigations in a family in which all its members exhibited xeroderma pigmentosum. PATIENTS AND METHODS: Since 1962, the father, mother, the 5 children and the maternal uncle were all followed-up in the dermatology department in Tunis for a variant of xeroderma pigmentosum. Clinical (dermatological, neurological and ophthalmologic), biological, photobiological and molecular biology investigations were carried out. RESULTS: Diagnosis of a variant of xeroderma pigmentosum was established on the delayed appearance (after the age of 4) of poikiloderma and the early onset of multiple carcinomas, without neurological disorders. Fifty-eight squamous cell and 3 basal cell carcinomas were diagnosed and treated by surgical exeresis or radiotherapy. The third child, treated with etretinate for 6 years, had developed 38 carcinomas. Contrary to the parents, whose first carcinomas had appeared at the age of 34 and 40 years, the cutaneous cancers in the children appeared early, between the ages of 17 and 24. The minimal erythematous dose was normal in all these patients. Conversely, the phototest revealed persistent erythema and the delayed appearance of multiple dyskeratosis cells. Molecular biology confirmed the diagnosis of xeroderma pigmentosum with the presence of a low level DNA repair. The third child, the father and the uncle respectively exhibited DNA repair rates of 32, 57 and 72%, compared with normal controls. The results of the complementarity tests conducted in the third child suggested that this family belonged to the genetic F group. Discussion The clinical and molecular data confirmed the diagnosis of xeroderma pigmentosum in this family and their genetic F group profile. However, this family exhibited clinical (the cutaneous involvement was more severe in the children) and molecular heterogeneity and the level of DNA repair was high in comparison with the levels (between 12 and 15%) reported by Japanese authors in group F xeroderma pigmentosum. The third child exhibited 10-fold more carcinomas that his siblings. This high rate of carcinoma may be explained by excessive exposure to sun and/or the retinoid treatment, particularly since his DNA repair rate (32%) was relatively high compared with that of severe (0-5%) and moderate (5-15%) forms of the disease.
Assuntos
Xeroderma Pigmentoso/diagnóstico , Xeroderma Pigmentoso/genética , Seguimentos , Humanos , LinhagemRESUMO
Xeroderma pigmentosum is an inheritable autosomal recessive DNA repair deficient syndrome characterized by a high predisposition to skin cancers. An elevated proportion of tumors from xeroderma pigmentosum patients harbor ultraviolet-induced mutations (CC:GG > TT:AA tandem transitions) of the p53 and/or the INK4a-ARF genes. Here, we report the clinical and molecular features of a 12 y old xeroderma pigmentosum patient who, in addition to severe cutaneous clinical symptoms, also had three unusual tumors, a mediastinal lymphoblastic lymphoma, an atypical fibroxanthoma, and an epithelioid hemangioma. Single strand conformation polymorphism and sequencing analysis of the p53 and INK4a-ARF genes were carried out in DNA from normal skin and different tumors (four actinic keratosis, two microinvasive squamous cell carcinomas, one basal cell carcinoma, and one atypical fibroxanthoma) from the patient. After characterization of the xeroderma pigmentosum C complementation group, we found unexpectedly that this patient also carried a germline mutation of the INK4a-ARF locus affecting the p16INK4A reading frame. Three different somatic mutations that all harbor the signature of ultraviolet light (two of p16INK4A and one of p53) were also detected in the basal cell carcinoma. We hypothesize that the germline mutation of p16INK4A, in association with the nucleotide excision repair defect, could explain the patient's unusual phenotype. Furthermore, this study confirms that concomitant somatic mutations of INK4a-ARF and p53 occur in some xeroderma pigmentosum associated tumors, and seem to accumulate during tumor progression rather than the initiation step.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/genética , Mutação em Linhagem Germinativa , Proteína Supressora de Tumor p14ARF/genética , Xeroderma Pigmentoso/genética , Carcinoma Basocelular/genética , Carcinoma de Células Escamosas/genética , Criança , Análise Mutacional de DNA , Reparo do DNA , Proteínas de Ligação a DNA/genética , Humanos , Queratinócitos/fisiologia , Ceratose/genética , Linfoma de Células T/genética , Masculino , Neoplasias Cutâneas/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
The RET proto-oncogene encodes a protein structurally related to transmembrane receptors with an intracellular tyrosine kinase domain. In human thyroid gland, the RET proto-oncogene is normally expressed in parafollicular C-cells. Thyroid C-cell hyperplasia is associated with inherited medullary thyroid carcinomas and is considered as a pre-neoplastic stage of C-cells disease. It has also been observed in thyroid tissues adjacent to follicular and papillary carcinomas. In order to study the relationship between a misfunctioning of the RET proto-oncogene and the presence of C-cell hyperplasia, we compared a series of thyroid glands presenting sporadic or radiation-associated tumours, as well as samples of unrelated normal thyroid tissues, for alteration in exons 10 and 11 of the gene and for the presence or absence of C-cell hyperplasia. Here we report a significantly higher frequency of C-cell hyperplasia present in peritumoural thyroid tissues of radiation-induced epithelial thyroid tumours, than in peritumoural of sporadic thyroid tumours or in control normal thyroid tissues (P=0.001). A G691S RET polymorphism was present with a higher frequency in radiation-induced epithelial thyroid tumours (55%) than in sporadic tumours (20%) and in control normal thyroid tissues (15%). Interestingly, this polymorphism was associated in the majority (88%) of radiation-induced tumours with a C-cell hyperplasia in the peritumoural tissues. Several explanations for this association are discussed.
Assuntos
Adenoma/genética , Carcinoma Papilar/genética , Proteínas de Drosophila , Neoplasias Induzidas por Radiação/genética , Polimorfismo de Nucleotídeo Único/genética , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/genética , Adenoma/etiologia , Adenoma/patologia , Adolescente , Adulto , Idoso , Carcinoma Papilar/etiologia , Carcinoma Papilar/patologia , Transformação Celular Neoplásica , Criança , Pré-Escolar , Códon , Relação Dose-Resposta à Radiação , Células Epiteliais/patologia , Células Epiteliais/efeitos da radiação , Feminino , Humanos , Hiperplasia , Linfócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Neoplasias Induzidas por Radiação/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Neoplasias da Glândula Tireoide/etiologia , Neoplasias da Glândula Tireoide/patologiaRESUMO
To understand the relationship between DNA repair, apoptosis, transcription, and cancer-proneness, we have studied the apoptotic response and the recovery of RNA synthesis following ultraviolet C and ultraviolet B irradiation in nucleotide excision repair deficient diploid fibroblasts from the cancer-prone xeroderma pigmentosum (XP) syndrome patients and the non-cancer-prone trichothiodystrophy (TTD) patients. Analysis of four XPD and four TTD/XPD fibroblast strains presenting different mutations on the XPD gene has shown that XPD cells are more sensitive to ultraviolet-induced apoptosis than TTD/XPD cells, and this response seems to be modulated by the type and the location of the mutation on the XPD gene. Moreover, the other xeroderma pigmentosum fibroblast strains analyzed (groups A and C) are more sensitive to undergo apoptosis after ultraviolet irradiation than normal human fibroblasts, showing that the cancer-proneness of xeroderma pigmentosum patients is not due to a deficiency in the ultraviolet-induced apoptotic response. We have also found that cells from transcription-coupled repair deficient XPA, XPD, TTD/XPD, and Cockayne's syndrome patients undergo apoptosis at lower ultraviolet doses than transcription-coupled repair proficient cells (normal human fibroblasts and XPC), indicating that blockage of RNA polymerase II at unrepaired lesions on the transcribed strand is the trigger. Moreover, XPD and XPA cells are more sensitive to ultraviolet-induced apoptosis than trichothiodystrophy and Cockayne's syndrome fibroblasts, suggesting that both cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone on the transcribed strand trigger apoptosis. Finally, we show that apoptosis is directly proportional to the level of inhibition of transcription, which depends on the density of ultraviolet-induced lesions occurring on transcribed sequences.
Assuntos
Apoptose/fisiologia , DNA Helicases , Reparo do DNA , Proteínas de Ligação a DNA , Predisposição Genética para Doença , Mutação/fisiologia , Proteínas/genética , Neoplasias Cutâneas/genética , Fatores de Transcrição , Raios Ultravioleta , Linhagem Celular , Anormalidades Congênitas/genética , Fibroblastos/efeitos da radiação , Cabelo/anormalidades , Humanos , Pele/patologia , Pele/fisiopatologia , Pele/efeitos da radiação , Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma PigmentosoRESUMO
The xeroderma pigmentosum group D (XPD) protein is a subunit of transcription factor TFIIH with DNA helicase activity. TFIIH has two functions, in basal transcription and nucleotide excision repair. Mutations in XPD that affect DNA repair but not transcription result in the skin cancer-prone disorder, xeroderma pigmentosum (XP). If transcription is also affected, the result is the multi-system disorder trichothiodystrophy (TTD), in which there is no skin cancer predisposition, or in rare cases, XP combined with Cockayne syndrome. Up till now there have been no reports of combined clinical features of XP and TTD. We have now identified two patients with some features of both these disorders. One of these, XP189MA, a 3-year-old girl with sun sensitivity, mental and physical developmental delay, has XPD mutations not previously reported, and barely detectable levels of nucleotide excision repair. The other, XP38BR, a 28-year-old woman with sun sensitivity, pigmentation changes and skin cancers typical of XP, has a mutation that has been identified previously, but only in TTD patients with no features of XP. The level of repair of UV damage in XP38BR is substantially higher than that in other patients with the same mutation. With both patients, polarized light microscopy revealed a 'tiger-tail' appearance of the hair, and amino acid analysis of the hair shafts show levels of sulfur-containing proteins intermediate between those of normal and TTD individuals. Our findings highlight the complexities of genotype-phenotype relationships in the XPD gene.
Assuntos
DNA Helicases , Proteínas de Ligação a DNA , Doenças do Cabelo/genética , Proteínas/genética , Fatores de Transcrição , Xeroderma Pigmentoso/genética , Adulto , Sequência de Aminoácidos , Apoptose/efeitos da radiação , Sequência de Bases , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Pré-Escolar , Análise Mutacional de DNA , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta à Radiação , Feminino , Doenças do Cabelo/patologia , Humanos , Mutação , Transtornos de Fotossensibilidade/genética , Transtornos de Fotossensibilidade/patologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Raios Ultravioleta , Xeroderma Pigmentoso/patologia , Proteína Grupo D do Xeroderma PigmentosoRESUMO
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the extracellular deposition of amyloid beta-peptide (Abeta) in the brain. Abeta is involved in the pathogenesis of AD but the molecular mechanisms of its neurotoxicity are unknown. Here, we report that Abeta exposure on human preneuronal NT2 cells provoked a strong and early up-regulation of growth arrest and DNA damage inducible gene (Gadd45 mRNA), an indicator of DNA damage and DNA excision-repair processes, strongly suggesting that Abeta causes an early DNA strand breakage leading to a cellular DNA repair response. Comet assay clearly demonstrated that both full-length Abeta (1-42), and its minimal cytotoxic fragment Abeta (25-35), caused DNA breakage as early as 3h after the start of Abeta exposure. This extensive DNA damage provoked by Abeta constitutes an early event in the pathogenic cascade leading to neuronal death which could contribute to the neuropathogenesis of AD.
Assuntos
Peptídeos beta-Amiloides/toxicidade , DNA/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/toxicidade , Biossíntese de Proteínas , Proteínas , Northern Blotting , Células Cultivadas , Ensaio Cometa , Dano ao DNA , Reparo do DNA , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fatores de Tempo , Regulação para Cima , Proteína bcl-X , Proteínas GADD45RESUMO
Murine gammaherpesvirus 72 (MHV-72) is a virus of wild rodents and serves as a convenient small animal model to understand the pathogenesis of Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV8) infection. In laboratory mice MHV-72 causes an acute infection of lung epithelial cells and establishes the latency in B lymphocytes. In this study, we investigated athymic nude and immunocompetent mice for distribution of virus in organs after infection with MHV-72. Ten days following subcutaneous dorsal injection of nude mice, virus replicated in lungs, lymphoid organs, salivary glands and also in mammary glands. The virus titre decreased by day 21 post-infection in former tissues, but increased in mammary glands. Presence of virus DNA sequences was detected in the lymphoid and non-lymphoid tissues until the death of the animals (about 1 month post-infection). Infection of immunocompetent mice with MHV-72 induced replication of virus up to 42 days post-infection in mammary glands reaching the highest level of infectious virus at day 8 post-infection. These data show that there is latent infection in mice never detected before. Moreover, virus DNA was detected using nested PCR (by amplification of a portion of gp150 gene sequence) in the mammary glands and the milk of mouse mothers infected with MHV-72 2 days before delivery. We demonstrated the presence of virus DNA also in the milk removed from the stomach of non-infected newborn mice, which were nourished by infected mothers (wet-nurses) for 1 or 2 days. The failure to detect the virus DNA in newborn mice lungs confirmed that they did not become infected from wet-nurses by the intranasal route. This suggests that MHV may be naturally transmitted to newborn mice via breast milk.