RESUMO
DICER1 is an endoribonuclease central to the generation of microRNAs (miRNAs) and short interfering RNAs (siRNAs). Germline mutations in DICER1 have been associated with a pleiotropic tumour predisposition syndrome and Wilms tumour (WT) is a rare manifestation of this syndrome. Three WTs, each in a child with a deleterious germline DICER1 mutation, were screened for somatic DICER1 mutations and were found to bear specific mutations in either the RNase IIIa (n = 1) or the RNase IIIb domain (n = 2). In the two latter cases, we demonstrate that the germline and somatic DICER1 mutations were in trans, suggesting that the two-hit hypothesis of tumour formation applies for these examples of WT. Among 191 apparently sporadic WTs, we identified five different missense or deletion somatic DICER1 mutations (2.6%) in four individual WTs; one tumour had two very likely deleterious somatic mutations in trans in the RNase IIIb domain (c.5438A>G and c.5452G>A). In vitro studies of two somatic single-base substitutions (c.5429A>G and c.5438A>G) demonstrated exon 25 skipping from the transcript, a phenomenon not previously reported in DICER1. Further we show that DICER1 transcripts lacking exon 25 can be translated in vitro. This study has demonstrated that a subset of WTs exhibits two 'hits' in DICER1, suggesting that these mutations could be key events in the pathogenesis of these tumours.
Assuntos
RNA Helicases DEAD-box/genética , Mutação em Linhagem Germinativa , Neoplasias Renais/genética , Ribonuclease III/genética , Tumor de Wilms/genética , Animais , Células COS , Pré-Escolar , Chlorocebus aethiops , Éxons , Feminino , Humanos , Neoplasias Renais/diagnóstico , Masculino , Mutação de Sentido Incorreto , Tumor de Wilms/diagnósticoRESUMO
We have examined the imprinting of the Wilms' tumour suppressor gene (WT1) in human tissues. We confirm that WT1 is biallelically expressed in the kidney, however, in five of nine preterm placentae WT1 was expressed largely or exclusively from the maternal allele. Monoallelic expression of WT1 was also found in two fetal brains. These data demonstrate that WT1 can undergo tissue specific imprinting. Furthermore, because monoallelic expression of WT1 was not found in all placentae examined, WT1 imprinting may be genetically polymorphic within the human population.
Assuntos
Genes do Tumor de Wilms , Mosaicismo , Polimorfismo Genético , Alelos , Sequência de Bases , Primers do DNA/genética , Feminino , Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Placenta/metabolismo , Gravidez , Distribuição TecidualRESUMO
We have examined the imprinting of the insulin-like growth factor II gene (IGF2) in ten normal kidney samples from children with renal embryonal neoplasms. In kidney samples from nine children with normal growth profiles, IGF2 mRNA was transcribed monoallelically, consistent with normal imprinting of the gene. But in one child who had generalized somatic overgrowth, IGF2 was transcribed from both alleles in her kidney, peripheral blood leukocytes and Wilms' tumour. These findings suggest that a defect in genomic imprinting can occur constitutionally, leading to growth abnormalities and predisposition to Wilms' tumour.
Assuntos
Genes do Tumor de Wilms , Gigantismo/genética , Fator de Crescimento Insulin-Like II/metabolismo , Neoplasias Renais/genética , Alelos , Humanos , Fator de Crescimento Insulin-Like II/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético , RNA Mensageiro/metabolismoRESUMO
Overexpression of the normally imprinted fetal insulin-like growth factor II (IGF2) has been implicated in the pathogenesis of the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). We have detected constitutional relaxation of imprinting of IGF2 in four children with somatic overgrowth who do not show diagnostic features of BWS. Three children showed constitutional abnormalities of H19 methylation. All four children showed nephromegaly and two developed Wilms' tumors. Gene methylation is known to be associated with gene silencing, and three children showed constitutional abnormalities of H19 gene methylation. Disruption of H19 methylation, and concomitant relaxation of IGF2 imprinting, provides another mechanism that can increase IGF2 expression in children with overgrowth. The accumulated data on normal and pathologic IGF2 expression are now sufficient to define an entity, "IGF2 overgrowth disorder," of which BWS may be one extreme manifestation. These findings have broad implications for the characterization of idiopathic overgrowth.
Assuntos
Síndrome de Beckwith-Wiedemann/genética , Transtornos do Crescimento/genética , Fator de Crescimento Insulin-Like II/genética , RNA não Traduzido , Sequência de Bases , Síndrome de Beckwith-Wiedemann/etiologia , Síndrome de Beckwith-Wiedemann/patologia , Criança , Pré-Escolar , Primers do DNA , Feminino , Expressão Gênica , Impressão Genômica , Transtornos do Crescimento/etiologia , Transtornos do Crescimento/metabolismo , Humanos , Lactente , Fator de Crescimento Insulin-Like II/metabolismo , Rim/patologia , Neoplasias Renais/etiologia , Neoplasias Renais/genética , Masculino , Metilação , Dados de Sequência Molecular , Mosaicismo , Proteínas Musculares/química , Proteínas Musculares/genética , Regiões Promotoras Genéticas , RNA Longo não Codificante , Tumor de Wilms/etiologia , Tumor de Wilms/genéticaRESUMO
BACKGROUND: We have recently reported an inverse relationship between colon cancer progression and tumour proliferative activity. Here, we extend our findings by evaluating the proliferative activity of liver metastatic lesions and primary colorectal cancers (CRC) that differ in their metastatic potential. METHODS: Using an earlier established multi-gene proliferation signature (GPS), proliferative levels were analysed in 73 primary CRCs and 27 liver metastases. RESULTS: Compared with primary CRCs, we observed a significantly lower expression of the GPS in liver metastases and confirmed their lower proliferative levels by quantitative RT-PCR and Ki-67 immunostaining. No difference could be detected in apoptotic indices as assessed by M30 immunostaining, indicating that the net growth rate is lower in metastases relative to primary tumours. Notably, relapsed primaries or those with established metastases had significantly lower proliferative activity than CRCs that were non-metastatic and did not relapse. CONCLUSION: Our results suggest that slow proliferation is a biological characteristic of both liver metastases and those primary tumours with the ability to metastasise. The delineation of the mechanisms underlying the inverse association between proliferation and CRC aggressiveness may be important for the development of new therapeutic strategies.
Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proliferação de Células , Neoplasias Colorretais/cirurgia , Perfilação da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Neoplasias Hepáticas/cirurgia , Estadiamento de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Wilms tumor is an embryonal kidney tumor involving complex pathology and genetics. The Wilms tumor locus on chromosome 11p13 is defined by the region of overlap of constitutional and tumor-associated deletions. Chromosome walking and yeast artificial chromosome (YAC) cloning were used to clone and map 850 kilobases of DNA. Nine CpG islands, constituting a "CpG island archipelago," were identified, including three islands that were not apparent by conventional pulsed-field mapping, and thus were at least partially methylated. Three distinct transcriptional units were found closely associated with a CpG island within the boundaries of a homozygous DNA deletion in a Wilms tumor.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Fosfatos de Dinucleosídeos , Genes do Tumor de Wilms/genética , Tumor de Wilms/genética , Passeio de Cromossomo , Sondas de DNA , Humanos , Transcrição GênicaRESUMO
Children with associated Wilms' tumor, aniridia, genitourinary malformations, and mental retardation (WAGR syndrome) frequently have a cytogenetically visible germ line deletion of chromosomal band 11p13. In accordance with the Knudson hypothesis of two-hit carcinogenesis, the absence of this chromosomal band suggests that loss of both alleles of a gene at 11p13 causes Wilms' tumor. Consistent with this model, chromosomes from sporadically occurring Wilms' tumor cells frequently show loss of allelic heterozygosity at polymorphic 11p15 loci, and therefore it has been assumed that allelic loss extends proximally to include 11p13. We report here that in samples from five sporadic Wilms' tumors, allelic loss occurred distal to the WAGR locus on 11p13. In cells from one tumor, mitotic recombination occurred distal to the gamma-globin gene on 11p15.5. Thus, allelic loss in sporadic Wilms' tumor cells may involve a second locus on 11p.
Assuntos
Aberrações Cromossômicas , Cromossomos Humanos Par 11 , Neoplasias Renais/genética , Tumor de Wilms/genética , Alelos , Criança , Pré-Escolar , Mapeamento Cromossômico , Feminino , Heterozigoto , Humanos , Lactente , Masculino , Modelos GenéticosRESUMO
Tumour cells have to undergo gene expression changes in order to metastasise and adapt to a new site. We investigated these changes in liver metastases of colorectal cancer by using genome-wide microarray analysis to profile the expression of 48 primary tumours and 28 liver metastases. Statistical analysis of these expression profiles using the significance analysis of microarrays (SAM) method identified 778 genes differentially expressed between primary tumours and metastases. Gene ontology analysis revealed that genes associated with tissue remodelling and immune response were upregulated in metastases relative to primary tumours, whereas genes associated with proliferation and oxidative phosphorylation were downregulated. Quantitative real-time PCR confirmed the differential expression of selected genes, osteopontin, versican, ADAM17, CKS2, PRDX1, CXCR4, CXCL12, and LCN2. The upregulation of genes associated with tissue remodelling and immune response are likely to be involved in metastatic invasion and colonisation of the new site because these genes can promote tumour progression. However, downregulation of genes associated with proliferation suggests that proliferation in metastases was reduced relative to primary tumours.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica , Genes Neoplásicos/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fosforilação Oxidativa , Regulação para CimaRESUMO
In situ hybridization was used to examine, in parallel, the localization of insulin-like growth factor II (Igf2) and WT1 transcripts in normal fetal kidney and Wilms' tumor. The expression of Igf2 and WT1 transcripts in the fetal kidney is almost complementary in both the epithelial and stromal cell lineages derived from the undifferentiated metanephrogenic blastema. The patterns of transcription of Igf2 in three Wilms' tumors appeared to be perturbed as compared to the normal fetal kidney. In these tumors Igf2 transcripts were detected in structures that are developmentally equivalent to the renal vesicle, which in the normal kidney do not contain Igf2 transcripts. These results suggest that Wilms' tumors arise from an alteration in the regulation of Igf2 mRNA synthesis.
Assuntos
Expressão Gênica , Genes do Tumor de Wilms , Fator de Crescimento Insulin-Like II/biossíntese , Neoplasias Renais/metabolismo , Rim/metabolismo , RNA Mensageiro/análise , Tumor de Wilms/metabolismo , Sequência de Bases , Feto , Idade Gestacional , Humanos , Hibridização In Situ , Fator de Crescimento Insulin-Like II/genética , Rim/citologia , Neoplasias Renais/genética , Neoplasias Renais/patologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Transcrição Gênica , Tumor de Wilms/genética , Tumor de Wilms/patologiaRESUMO
Wilms' tumor (WT), a childhood cancer of the kidney, occurs in both familial and sporadic forms. Chromosome 11 genes have been implicated in the etiology of WT, and mutations in a gene at chromosomal band 11p13, WT1, have been identified in a few WT cases. However, 11p13 has been excluded as the site of the predisposition mutation segregating in several large WT families, which implies the existence of a non-11p familial predisposition gene. Recently, loss of heterozygosity for 16q markers located between chromosomal bands 16q13 and 16q22 has been reported in approximately 20% of sporadic Wilms' tumors. To determine if this region of 16q harbors the non-11p familial WT gene, a genetic linkage study of five WT families was undertaken. Using multipoint analyses, we ruled out genetic linkage of familial WT predisposition to 16q.
Assuntos
Cromossomos Humanos Par 16 , Genes do Tumor de Wilms , Neoplasias Renais/genética , Escore Lod , Tumor de Wilms/genética , Bandeamento Cromossômico , Feminino , Marcadores Genéticos , Humanos , Masculino , Linhagem , Polimorfismo de Fragmento de RestriçãoRESUMO
Mouse p57(Kip2) arrests cells in G1 by functioning as a strong inhibitor of several G1 cyclin/Cdk complexes (Lee et al., 1995; Matsuoka et al., 1995; Sherr and Roberts, 1995). Human p57(KIP2) has been suggested to be a tumour suppressor gene because of its location at 11p15.5 which frequently undergoes maternal allele LOH in several types of cancer (Matsuoka et al., 1995; Sherr and Roberts, 1995; Hatada and Mukai, 1995). This suggestion was supported by the discovery that mouse p57(Kip2) is imprinted with expression from only the maternally inherited allele (Hatada and Mukai, 1995). Interestingly, p57(KIP2) is several hundred kilobases from the imprinted H19 and IGF2 genes which are involved in growth regulation (Hoovers et al., 1995). Here we show that human p57(KIP2) is imprinted with expression from the maternal allele. However, unlike the mouse, the imprinting is incomplete with significant expression from the paternal allele depending on the tissue examined. We have also shown that the imprinting of p57(KIP2) occurs independently of the H19/IGF2 domain and thus there must be at least two imprinted domains in 11p15.5. Finally, by examining Wilms tumours we have shown that following maternal 11p LOH, p57(KIP2) was expressed from the paternal allele. Therefore, p57(KIP2) cannot function as an imprinted tumour suppressor gene, at least in Wilms tumour.
Assuntos
Cromossomos Humanos Par 11 , Genes Supressores de Tumor , Impressão Genômica , Proteínas Nucleares/genética , Tumor de Wilms/genética , Adulto , Alelos , Criança , Inibidor de Quinase Dependente de Ciclina p57 , Feminino , Deleção de Genes , Expressão Gênica , Heterozigoto , Humanos , Masculino , Proteínas Nucleares/biossíntese , Reação em Cadeia da Polimerase , Transcrição Gênica , Tumor de Wilms/metabolismoRESUMO
WT1 is a tumor suppressor gene that has been implicated in Wilms tumor, and is expressed in cells of mesodermal origin. The Wit-1 gene is located approximately 2 kb from the WT1 gene, and is expressed coordinately with WT1. WT1 and Wit-1 are bi-directionally transcribed from the same promoter region. We have screened a human fetal kidney cDNA library to identify novel WT1 cDNA clones. Here we report the cloning of cDNA clones which span part of intron 1 of WT1, exon 1, upstream sequences between WT1 and Wit-1 and part of the Wit-1 gene. Northern blot and RNAase protection analysis using subcloned fragments of the cDNAs corresponding to regions from within intron 1 of WT1 suggest that a 7-10 Kb RNA is expressed in human fetal kidney, which overlaps with WT1 and is transcribed in the same direction as Wit-1.
Assuntos
Genes do Tumor de Wilms , Oligonucleotídeos Antissenso/genética , Transcrição Gênica , Sequência de Bases , Clonagem Molecular , DNA Complementar , Humanos , Íntrons , Rim/embriologia , Rim/metabolismo , Dados de Sequência MolecularRESUMO
WT1 is a tumour suppressor gene expressed in a specific temporal and spatial pattern in the developing kidney. Up to 15% of Wilms tumours have point mutations in the WT1 gene coding sequence. We have now investigated whether mutations in the WT1 promoter could be associated with loss of control WT1 expression and subsequent Wilms tumour formation. Using single-strand conformational polymorphism (SSCP) analysis we analysed 39 sporadic Wilms tumours for WT1 promoter mutations. We found six linked common sequence polymorphisms and two unlinked less frequent polymorphisms which allowed us to identify four tumours with loss of heterozygosity but none with point mutations, small deletions, insertions or rearrangements. We therefore conclude that WT1 promoter mutations are unlikely to play an important role in Wilms tumorigenesis.
Assuntos
Genes do Tumor de Wilms , Neoplasias Renais/genética , Mutação , Regiões Promotoras Genéticas , Tumor de Wilms/genética , Sequência de Bases , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
Relaxation of IGF2 imprinting occurs in Wilms tumours and many other cancers, but the mechanism of loss of imprinting (LOI) remains unknown. To investigate the role of altered DNA methylation in LOI, we examined the pattern of methylation of the human insulin-IGF2 region in Wilms tumours and the normal kidney. The analysis included regions homologous to three 'differentially methylated regions' of the mouse Igf2 gene (dmrs 0, 1 and 2). In tumours displaying normal IGF2 imprinting, and in the normal kidney, maternal allele-specific DNA methylation was identified spanning exons 2 and 3. This region is homologous to dmr 0, a site of maternal-specific differential methylation in the mouse. In Wilms tumours with relaxed imprinting or 11p15.5 LOH this region was unmethylated. No other differential methylation was identified. In particular, two sites of paternal methylation in the mouse (dmrs 1 and 2), and all three imprinted IGF2 promoters were not methylated in the kidney or in Wilms tumours. We postulate that LOI in Wilms tumours is associated with loss of maternal allele-specific methylation from a region located upstream of the imprinted IGF2 promoters. This region may contain cis acting sequences that coordinately influence imprinting.
Assuntos
Fator de Crescimento Insulin-Like II/genética , Neoplasias Renais/genética , Tumor de Wilms/genética , Animais , Southern Blotting , Metilação de DNA , Desoxirribonuclease HpaII/genética , Desoxirribonuclease HpaII/metabolismo , Éxons , Impressão Genômica , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Rim/fisiologia , Camundongos , Dados de Sequência Molecular , Sequências Reguladoras de Ácido NucleicoRESUMO
The specificity of IGF2 promoter imprinting was examined in embryonal tissues and Wilms tumour. In several fetal tissues of approximately 12 weeks gestation, IGF2 was found to be monoallelically expressed from all IGF2 promoters i.e. P1, P2, P3 and P4. However, in tissues of slightly older gestation age (15-17 weeks) relaxation of imprinting at the P1 promoter was evident, although the P2-P4 promoters remained imprinted. These data indicate that early in embryogenesis a population of cells exists in which all IGF2 promoters are imprinted, but that as development proceeds the imprinting of the P1 promoter is relaxed. The pattern of IGF2 promoter imprinting was also analysed in Wilms tumour. In some tumours, the pattern of promoter imprinting was identical to that found in early fetal kidney, indicating that this tumour originates within early embryonic kidney tissue. In contrast, in tumours in which relaxation of imprinting had occurred, imprinting relaxation affected all IGF2 promoters. This aberrant pattern of promoter imprinting, which was not detected in fetal kidney, provides further evidence that pathological relaxation of IGF2 imprinting is involved in the genesis of Wilms tumour.
Assuntos
Deleção Cromossômica , Cromossomos Humanos Par 11 , Desenvolvimento Embrionário e Fetal , Fator de Crescimento Insulin-Like II/genética , Neoplasias Renais/embriologia , Neoplasias Renais/genética , Regiões Promotoras Genéticas , Tumor de Wilms/embriologia , Tumor de Wilms/genética , Adulto , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Éxons , Feminino , Humanos , Rim/embriologia , Rim/patologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético , Gravidez , Transcrição GênicaRESUMO
BACKGROUND: Loss of imprinting (LOI) of the H19/IGF2 domain is a common feature of Wilms tumour. The GTL2/DLK1 domain is also imprinted and is structurally similar to H19/IGF2. The question arises as to whether DLK1 also undergoes LOI in Wilms tumour, or whether the LOI mechanism is restricted to the H19/IGF2 domain. AIM: To investigate the imprinting status of DLK1 in Wilms tumours with IGF2 LOI. The cellular localisation of DLK1 in the tumours was also examined. METHODS: DLK1 expression was measured by quantitative real time polymerase chain reaction (Q-PCR) in 30 Wilms tumours that had previously been classified according to whether they had IGF2 LOI, WT1 mutations, or 11p15.5 loss of heterozygosity. Allele specific expression of DLK1 was examined by direct sequencing using a DLK1 exon 5 polymorphism (rs1802710). Immunohistochemical analysis of DLK1 was performed on 13 tumours and two intralobar nephrogenic rests, in addition to two fetal kidneys and one fetal skeletal muscle sample. RESULTS: Ten of 30 tumours were heterozygous for rs1802710 and all tumours showed retention of imprinting of DLK1. Moderate to high expression of DLK1 was detected by Q-PCR in nine of 13 tumours with myogenic differentiation. Immunohistochemical expression of DLK1 was detected in the myogenic elements. CONCLUSION: LOI does not occur at the GTL2/DLK1 domain in Wilms tumour. This finding suggests that LOI at 11p15.5 does not reflect non-specific disruption of a shared imprinting mechanism. DLK1 expression in Wilms tumour might reflect the presence of myogenic differentiation, rather than an alteration of its imprinting status.
Assuntos
Impressão Genômica/genética , Glicoproteínas/genética , Neoplasias Renais/genética , Proteínas de Neoplasias/genética , Tumor de Wilms/genética , Alelos , Epigênese Genética/genética , Genes do Tumor de Wilms , Heterozigoto , Humanos , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intracelular , Rim/química , Rim/embriologia , Neoplasias Renais/patologia , Proteínas de Membrana/genética , Polimorfismo Genético/genética , Proteínas/genética , RNA Longo não Codificante , RNA Neoplásico/análise , Tumor de Wilms/patologiaRESUMO
"Genetic mosaicism" describes the presence of two or more populations of cells within a single individual that differ in their genomic constitution. Although the occurrence of asymmetric overgrowth in Wiedemann-Beckwith syndrome (WBS) suggests that mosaicism has some role in the WBS phenotype, no direct evidence for this has been published. WBS is a congenital overgrowth syndrome with variable phenotype linked to the imprinted gene cluster on chromosome region 11p15. We have performed a molecular survey of multiple organs and tissues in a case of WBS with a high degree of mosaic paternal 11p15 uniparental disomy (UPD). The organs most severely affected were those with the highest percentage of cells with UPD. In particular there was a striking difference in the degree of mosaicism for 11p15 UPD between the extremely enlarged left adrenal and non-enlarged right adrenal gland. This result indicates that the proportion of paternal 11p15 UPD cells correlates with the tissue phenotype of WBS. Our results suggest that high proportions of abnormal cells result from a combination of stochastic events and cell selection. Mosaicism may explain the variable phenotypes including hemihyperplasia and predisposition to childhood cancers in WBS patients.