Identification of protein-coding and intronic noncoding RNAs down-regulated in clear cell renal carcinoma

The clear cell subtype of renal cell carcinoma (RCC) is the most lethal and prevalent cancer of the urinary system. To investigate the molecular changes associated with malignant transformation in clear cell RCC, the gene expression profiles of matched samples of tumor and adjacent non-neoplastic tissue were obtained from six patients. A custom-built cDNA microarray platform was used, comprising 2292 probes that map to exons of genes and 822 probes for noncoding RNAs mapping to intronic regions. Intronic transcription was detected in all normal and neoplastic renal tissues. A subset of 55 transcripts was significantly down-regulated in clear cell RCC relative to the matched nontumor tissue as determined by a combination of two statistical tests and leave-one-out patient cross-validation. Among the down-regulated transcripts, 49 mapped to untranslated or coding exons and 6 were intronic relative to known exons of protein-coding genes. Lower levels of expression of SIN3B, TRIP3, SYNJ2BP and NDE1 (P<0.02), and of intronic transcripts derived from SND1 and ACTN4 loci (P<0.05), were confirmed in clear cell RCC by Real-time RT-PCR. A subset of 25 transcripts was deregulated in additional six nonclear cell RCC samples, pointing to common transcriptional alterations in RCC irrespective of the histological subtype or differentiation state of the tumor. Our results indicate a novel set of tumor suppressor gene candidates, including noncoding intronic RNAs, which may play a significant role in malignant transformations of normal renal cells. (C) 2008 Wiley-Liss, Inc.

DNA methylation in the CTCF-binding site I and the expression pattern of the H19 gene: Does positive expression predict poor prognosis in early stage head and neck carcinomas?

Loss of allele-specific expression by the imprinted genes IGF2 and H19 has been correlated with a differentially methylated region (DMR) upstream to the H19 gene. The H19-DMR contains seven potential CCCTC-binding factor (CTCF) binding sites. CTCF is a chromatin insulator and a multifunctional transcription factor whose binding to the H19-DMR is suppressed by DNA methylation. Our study included a group of 41 head and neck squamous cell carcinoma (HNSCC) samples. The imprinting status of the H19 gene was analyzed in 11 out of 35 positive cases for H19 gene expression, and only 1 of them showed loss of imprinting. We detected a significant correlation (P=0.041, Fisher's exact test) between H19 expression and tumor recurrence. Among H19 positive cases, six were T2, in which five developed recurrence and/or metastasis. Inversely, in the group of tumors that showed no H19 gene expression, 5 out of 24 were T2 and only I presented regional recurrence. These data support the hypothesis that H19 expression could be used as a prognostic marker to indicate recurrence in early stage tumors. We also examined the methylation of the CTCF binding site 1 in a subgroup of these samples. The H19 gene silencing and loss of imprinting were not correlated with the methylation pattern of the CTCF binding site 1. However, the significant correlation between H19 expression and tumor recurrence suggest that this transcript could be a marker for the progression of HNSCC. (c) 2005 Wiley-Liss, Inc.

CDHI promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer

Background: the E-cadherin gene (CDH1) maps, at chromosome 16q22.1, a region often associated with loss of heterozygosity (LOH) in human breast cancer. LOH at this site is thought to lead to loss of function of this tumor suppressor gene and was correlated with decreased disease-free survival, poor prognosis, and metastasis. Differential CpG island methylation in the promoter region of the CDH1 gene might be an alternative way for the loss of expression and function of E-cadherin, leading to loss of tissue integrity, an essential step in tumor progression.Methods: the aim of our study was to assess, by Methylation-Specific Polymerase Chain Reaction (MSP), the methylation pattern of the CDH1 gene and its possible correlation with the expression of E-cadherin and other standard immunohistochemical parameters (Her-2, ER, PgR, p53, and K-67) in a series of 79 primary breast cancers ( 71 infiltrating ductal, 5 infiltrating lobular, 1 metaplastic, 1 apocrine, and 1 papillary carcinoma).Results: CDH1 hypermethylation was observed in 72% of the cases including 52/71 ductal, 4/5 lobular carcinomas and 1 apocrine carcinoma. Reduced levels of E-cadherin protein were observed in 85% of our samples. Although not statistically significant, the levels of E-cadherin expression tended to diminish with the CDH1 promoter region methylation. In the group of 71 ductal cancinomas, most of the cases of showing CDH1 hypermethylation also presented reduced levels of expression of ER and PgR proteins, and a possible association was observed between CDH1 methylation and ER expression ( p = 0.0301, Fisher's exact test). However, this finding was not considered significant after Bonferroni correction of p-value.Conclusion: Our preliminary findings suggested that abnormal CDH1 methylation occurs in high frequencies in infiltrating breast cancers associated with a decrease in E-cadherin expression in a subgroup of cases characterized by loss of expression of other important genes to the mammary carcinogenesis process, probably due to the disruption of the mechanism of maintenance of DNA methylation in tumoral cells.

Aspectos moleculares do câncer tiroideano

A proliferação da célula tiroideana normal é regulada por fatores de crescimento estimuladores e inibidores, que atuam através de seus receptores de membrana e, subseqüentemente, através de transdutores citoplasmáticos. Na glândula normal adulta, o equilíbrio de sinais é tal que a proliferação é mínima, enquanto nas neoplasias o crescimento resulta de um distúrbio irreversível desse equilíbrio. Apesar do número de moléculas envolvidas nesse processo ser grande, apenas um pequeno subgrupo parece estar envolvido na tumorigênese tiroideana. Tais proteínas são codificadas pelos genes RAS, RET, NTRK1 e TP53. O transdutor de sinais ras é ativado por mutações em ponto e constitui uma alteração genética precoce nos tumores com histologia folicular. Os genes dos receptores de crescimento RET e NTRK1 são alterados por rearranjos cromossômicos do tipo translocação ou inversão nos carcinomas papilares e por mutações em ponto nos medulares. As alterações do gene TP53, por sua vez, têm sido observadas em carcinomas tiroideanos pobremente diferenciados e na maioria dos indiferenciados, o que sugere sua participação na progressão dessas lesões. O modelo molecular da carcinogênese tiroideana, embora ainda incompleto, pode fornecer instrumentos importantes para o diagnóstico diferencial e para o desenvolvimento de novas técnicas terapêuticas nesse grupo de neoplasias.

Alterations of the TP53 Gene in Gastric and Esophageal Carcinogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Loss of imprinting and loss of heterozygosity on 11p15.5 in head and neck squamous cell carcinomas

Background. IGF2 and H19 are reciprocal imprinted genes with paternal and maternal monoallelic expression, respectively. This is interesting, because IGF2 is known as a growth factor, and H19 encodes a RNA with putative tumor suppressor action. Furthermore, IGF2 and H19 are linked genes located on chromosome 11p15.5, a common site of loss of heterozygosity in human cancers.Methods. We performed an allelic-typing assay using a PCR-RFLP-based method for identification of heterozygous Informative cases in head and neck squamous cell carcinomas. Tumoral total RNA was extracted from each of the heterozygotes and further studied by RT-PCR analysis.Results. We detected the expression of the IGF2 gene in 10 of 10 informative cases. Two cases exhibited LOI of the IGF2 gene as evidenced by biallelic expression, and in another case, LOH was coupled with monoallelic expression of this growth factor. LOI for the H19 gene was observed in 1 of 14 informative samples analyzed. In this case, we also detected parallel mono-allelic expression of the IGF2 gene. Down-regulation of the H19 gene was observed in 10 of 14 cases.Conclusion. These findings support the hypothesis that H19 may be a tumor suppressor gene involved In head and neck carcinogenesis. Furthermore, our data showed that genetic and epigenetic chances at 11p15.5 could lead to abnormal expression of imprinted genes in HNSCC. (C) 2001 John Wiley & Sons, Inc.

Frequent p53 mutations and occasional loss of chromosome 4 in invasive bladder carcinoma induced by N-butyl-N-(4-hydroxybutil)nitrosamine in B6D2F1 mice

B6D2F1 mice (45/group) were treated with N-butyl-N-(4- hydroxybutyl)nitrosamine (BBN) or uracil as follows: Group 1 received 0.05% BBN in drinking water for the entire experiment, Group 2 received 5 mg of BBN by gastric gavage in 0.1 mL of 20% ethanol twice per week for 10 wk, Group 3 received a 2.5% uracil-containing diet for the entire experiment, and Group 4 was controls (received 0.1 mL of 20% ethanol by gavage twice per week for 10 wk). The surviving mice in Group 1 were killed after week 26 and those in the other groups after week 30. By week 15, three of 11 Group 1 and one of 15 Group 2 mice had bladder carcinoma. By 26 and 30 wk, respectively, invasive carcinomas were observed in 33 of 34 and six of 21 mice in Groups 1 and 2 and renal pelvic carcinomas in 11 of 34 and three of 21 mice in Groups 1 and 2. Four of 19 uracil-treated mice had bladder nodular hyperplasia. By polymerase chain reaction-single-strand conformation polymorphism and sequence analyses, 16 of 20 and two of five bladder carcinomas from Groups 1 and 2, respectively, showed mutations in the p53 gene. Ha-ras mutation was present in one case. Loss of heterozygosity analysis with simple-sequence length polymorphism markers for chromosome 4 showed that 10 of 21, two of 15, and nine of 13 mice in Groups 1-3, respectively, had heterozygous or homozygous deletions. B6D2F1 mice are therefore susceptible to the urothelial carcinogenic effects of BBN and develop frequent p53 mutations and chromosome 4 deletions. Chromosome 4 deletions were also seen with uracil.

Blepharocheilodontic (BCD) syndrome: Expanding the phenotype?

We describe affected individuals in three generations of a family and another sporadic case, all Brazilian patients, with a combination of signs that diagnose the BCD syndrome. In addition to the cardinal signs, the sporadic case has hypothyroidism and imperforate anus, which was observed previously in one patient. The broadened phenotype and the possibility of involvement of p63 and IRF6 genes in this condition are discussed. © 2003 Wiley-Liss, Inc.

Genetic alterations in benign lesions: Chronic gastritis and gastric ulcer

Aim: To investigate the occurrence of chromosome 3, 7, 8, 9, and 17 aneuploidies, TP53 gene deletion and p53 protein expression in chronic gastritis, atrophic gastritis and gastric ulcer, and their association with H pylori infection. Methods: Gastric biopsies from normal mucosa (NM, n = 10), chronic gastritis (CG, n = 38), atrophic gastritis (CAG, n = 13) and gastric ulcer (GU, n = 21) were studied using fluorescence in situ hybridization (FISH) and immunohistochemical assay. A modified Giemsa staining technique and PCR were used to detect H pylori. An association of the gastric pathologies and aneuploidies with H pylori infection was assessed. Results: Aneuploidies were increasingly found from CG (21%) to CAG (31%) and to GU (62%), involving mainly monosomy and trisomy 7, trisomies 7 and 8, and trisomies 7, 8 and 17, respectively. A significant association was found between H pylori infection and aneuploidies in CAG (P = 0.0143) and GU (P = 0.0498). No TP53 deletion was found in these gastric lesions, but p53-positive immunoreactivity was detected in 45% (5/11) and 12% (2/17) of CG and GU cases, respectively. However, there was no significant association between p53 expression and H pylori infection. Conclusion: The occurrence of aneuploidies in benign lesions evidences chromosomal instability in early stages of gastric carcinogenesis associated with H pylori infection, which may confer proliferative advantage. The increase of p53 protein expression in CG and GU may be due to overproduction of the wild-type protein related to an inflammatory response in mucosa. © 2006 The WJG Press. All rights reserved.

Quantitative cell-cycle protein expression in oral cancer assessed by computer-assisted system

The knowledge of cell-cycle control has shown that the capacity of malignant growth is acquired by the stepwise accumulation of defects in specific genes regulating cell growth. Histologic diagnosis might be improved by a quantitative evaluation of more specific diagnosis biomarkers, which could help to precisely identify pre-malignant and malignant oral lesions. The aim of the present study is to evaluate whether computer-based quantitative assessment of p53, PCNA and Ki-67 immunohistochemical expression, could be used clinically to foresee the risk of oral malignant transformation. This retrospective study was carried out in ninety-five oral biopsies, 27 were classified as fibrous inflammatory hyperplasia, 40 as leukoplakia and 28 as oral squamous cell carcinoma. Sixteen out of the 40 leukoplakia were diagnosed as non-dysplastic leukoplakia, the other 24 being dysplastic leukoplakia, of which 50.0% were classified as moderate to severe dysplasia. Comparison of the four groups of oral tissues showed significant rises in p53 and Ki-67 positivity index, which increased steadily in the order benign, pre-malignant, and malignant. In contrast, it was not possible to relate higher PCNA levels with pre-malignant and malignant oral lesions. We therefore conclude that PCNA immunohistochemistry expression is probably an inappropriate marker to identify oral carcinogenesis, whereas joint quantitative evaluation of p53 and Ki-67, appears to be useful as a tumor marker, providing a pre-diagnostic estimate of the potential for cell-cycle deregulation of the oral proliferate status.

Análise mutacional da região dos exons 5 a 8 do gene supressor de tumor p53 em neoplasias mamárias caninas

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Padrão de metilação dos genes CDH1, BRCA1, hMLH1 e polimorfismos das enzimas TS e MTHFR do ciclo do folato em tecido tumoral mamário

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Investigação citogenética na síndrome mielodisplásica infantil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Análise citogenética e molecular do gene FOXO3 em síndrome mielodisplásica

Pós-graduação em Genética - IBILCE

Análise da expressão de miRNAs em carcinoma hepatocelular

O carcinoma hepatocelular corresponde à neoplasia maligna primária mais comum do fígado e ao quinto tumor sólido mais frequente no mundo. Altamente letal, permanece como um grave problema de saúde pública em virtude das dificuldades no diagnóstico precoce e na elaboração de medidas terapêuticas efetivas. Estudos recentes no ramo da biologia molecular sugerem que o perfil de miRNAs no carcinoma hepatocelular pode influir consideravelmente na identificação de fatores de riscos associados a oncogenes ou genes supressores. Objetivou-se avaliar a expressão de miRNA 135b, miRNA 181a-5p e miRNA 181a-3p em amostras de Carcinoma Hepatocelular e de Hepatite C Crônica e correlacioná-las de maneira a buscar prováveis biomarcadores relacionados ao mecanismo de carcinogenese. A investigação foi feita em seis pacientes com carcinoma hepatocelular e vinte e quatro casos de Hepatite C Crônica, procedentes do Pará, Norte do Brasil. Todas as amostras de Carcinoma Hepatocelular foram submetidas à microdissecção, para posterior extração do RNA. Para a extração do RNA total e do microRNA foi utilizado o kit AllPrep DNA/RNA FFPE Kit (Qiagen), quantificados pelo equipamento Qubit® 2.0 Fluorometer (Invitrogen) para concentração padrão final de 5ng/μL. Em seguida cDNA foi obtido, utilizando-se TaqMan® MicroRNA Reverse Transcription(AppliedBiosystems). As análises estatísticas foram realizadas nos softwares SPSS 17.0, usando o teste de Mann-Whitney, considerando como significantes valores de p<0,05. Os resultados demonstraram diferenças significativas dos níveis de expressão do miR181a-3p e do miR181a-5p no carcinoma hepatocelular (médias 3,94 e 17,9, respectivamente) em relação à hepatite C crônica (médias de 1,18 e 1,8, respectivamente) com P valor de 0,005 e 0,003. Nesse estudo, observou-se que os miRNAs 181a-3p e 181a-5p, especialmente o 181a-5p, foram significativamente mais expressos nas amostras de carcinoma hepatocelular, quando comparados ao tecido hepático não tumoral com hepatite C crônica. Portanto, os microRNAs possuem características interessantes que os favorecem como possíveis marcadores biológicos no rastreamento de tumores para diagnóstico precoce e terapias alvo selecionadas.