RESUMO
METHODS AND RESULTS: By differential display we isolated a new cDNA-fragment, named C13, that is downregulated in malignant prostate tissues. Northern hybridization revealed the fragment to be part of 3.0 and 4.4 kb mRNAs. Fluorescence in situ hybridization, Southern blotting and radiation hybrid mapping demonstrated a chromosomal localization of C13 on 13q12-14 closest to the SHGC-34125 marker. In the 5% chromosomal environment of C13 we detected changes of the allelic status in 13 of 21 prostate cancers. A downregulation was detected at the mRNA level in patients with advanced carcinoma. The 3.0 kb full length cDNA clone encodes a protein with an open reading frame of 2,202 bp or 733 amino acids. The corresponding protein contains a putative nuclear localization signal, several glutamine clusters and an alpha-helix-rich domain. By in situ RNA hybridization we could demonstrate the mainly epithelial expression of the C13 mRNA in prostatic tissue. CONCLUSIONS: The localization of C13 between the tumor suppressor genes BRCA-2 and RB-1, the detected allelic imbalances, the downregulation of its mRNA in some prostatic cancer tissues, the epithelial expression and the described protein structure suggest that this gene encodes a protein that may have tumor or metastasis suppressing function in prostate tissue.
Assuntos
Cromossomos Humanos Par 13/genética , DNA de Neoplasias/genética , Proteínas de Neoplasias/genética , Neoplasias da Próstata/genética , Proteína do Retinoblastoma/genética , Fatores de Transcrição/genética , Proteína BRCA2 , DNA Complementar/genética , DNA Complementar/isolamento & purificação , DNA Complementar/metabolismo , DNA de Neoplasias/isolamento & purificação , DNA de Neoplasias/metabolismo , Perfilação da Expressão Gênica , Genes Supressores de Tumor , Humanos , Hibridização in Situ Fluorescente , Perda de Heterozigosidade , Masculino , Proteínas de Neoplasias/química , Neoplasias da Próstata/química , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Mapeamento de Híbridos Radioativos , Técnica de Amplificação ao Acaso de DNA Polimórfico , Proteína do Retinoblastoma/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/químicaRESUMO
A four-step procedure for the efficient and systematic mining of whole EST libraries for differentially expressed genes is presented. After eliminating redundant entries from the EST library under investigation (step 1), contigs of maximal length are built upon each remaining EST using about 4 000 000 public and proprietary ESTs (step 2). These putative genes are compared against a database comprising ESTs from 16 different tissues (both normal and tumour affected) to determine whether or not they are differentially expressed (step 3; electronic northern). Fisher's exact test is used to assess the significance of differential expression. In step 4, an attempt is made to characterise the contigs obtained in the assembly through database comparison. A case study of the CGAP library NCI_CGAP_Br1.1, a library made from three (well, moderately, and poorly differentiated) invasive ductal breast tumours (2126 ESTs in total) was carried out. Of the maximal contigs, 139 were found to be significantly (alpha = 0.05) over-expressed in breast tumour tissue, while 13 appeared to be down-regulated.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Etiquetas de Sequências Expressas , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos/genética , Animais , Northern Blotting/métodos , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Biologia Computacional , Bases de Dados Factuais , Regulação para Baixo , Humanos , Mitocôndrias/genética , Invasividade Neoplásica , RNA Mensageiro/análise , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Ribossomos/genética , Homologia de Sequência do Ácido Nucleico , Software , Estatística como AssuntoRESUMO
Microarrays are a powerful tool in modern genome analysis. The massive parallel analysis of the RNA expression level of thousands of genes accelerates the research in molecular biology and leads to new insights in signalling pathways. Furthermore, genomic gains or losses can be analyzed for complete genomes in a single experiment. This creates new opportunities for understanding distinct chromosomal changes in the development of cancer. The ability to genotype patients using single nucleotide polymorphisms is of primordial interest to the pharmaceutical industry and may result in a more individualized medicine. Tissue microarrays are very useful in the investigation of the expression of a particular gene in hundreds of specimens by in situ hybridization. Also, protein microarrays are currently being developed for studying protein-protein interactions. In conclusion, microarrays will revolutionize all aspects of molecular biology, and will probably have the same impact as the polymerase chain reaction.
Assuntos
Perfilação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise Serial de Proteínas/métodos , Análise Serial de Tecidos/métodos , Animais , Genômica/métodos , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Prostate cancer (CaP) is one of the most common neoplasms in the USA and Europe. We used differential display PCR (DD-PCR) to identify genes related to the development of prostate cancer. METHODS: The RNA of 4 patients with untreated CaP was analyzed for differentially expressed genes. Using DD-PCR, we identified a downregulated cDNA-fragment in these prostate cancer cells. This fragment (N7) was cloned and further analyzed by CMRT-PCR, Northern-blot analysis, and in situ hybridization. RESULTS: Sequence analysis revealed that N7 is identical to the 3'-untranslated region of the recently described immediate early gene Cyr61. Comparative multiplex RT-PCR with sequence specific primers showed that Cyr61 is downregulated in the tumor tissue of 7 out of 13 patients. By in situ hybridization we could demonstrate that the expression of Cyr61 is restricted to the epithelium of the prostate. Immunohistochemical analysis showed that Cyr61 protein could be found in the epithelium of normal prostatic tissue, whereas prostate cancer tissue showed a marked decrease of Cyr61 protein expression. CONCLUSIONS: Cyr61 is a member of the emerging family of extracellular signaling proteins and enhances the effect of bFGF. The changed pattern of expression Cyr61 might therefore contribute to the altered interactions between epithelial and stromal cells in prostate carcinoma.
Assuntos
Regulação Neoplásica da Expressão Gênica , Substâncias de Crescimento/genética , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular , Neoplasias da Próstata/metabolismo , Transdução de Sinais , Animais , Sequência de Bases , Northern Blotting , Proteína Rica em Cisteína 61 , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Masculino , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , DNA Polimerase Dirigida por RNA , Alinhamento de Sequência , Células Tumorais CultivadasRESUMO
OBJECTIVES: The aim of this study was to investigate whether a significant difference consists between patients with and without prostate carcinoma (CaP) regarding the ratio of prostate-specific antigen (PSA) in complex with major proteinase inhibitors to noncomplexed (free) PSA (FPSA). METHODS: We analyzed the sera of 29 patients with untreated CaP, 34 patients with benign prostatic hyperplasia (BPH), and 33 men with no symptoms of prostate disease for the amount of FPSA and total PSA (TPSA) with the Immulite chemiluminescent enzyme immunometric assay. RESULTS: FPSA was found only as a minor fraction in all sera tested. Calculation of the percentage of FPSA from TPSA revealed a significant difference between patients with CaP (median, 9.55%) versus patients with BPH (median, 17.07%; P = 0.00001) or versus healthy men (median, 16.11%; P = 0.0006). Considering only patients with PSA values less than 10 ng/mL, the difference between patients with CaP versus patients with BPH remained significant (P = 0.026). The specificity for differentiation between CaP and BPH at a sensitivity level of 89% for the combined evaluation of the proportion of FPSA and TPSA increased from 30% (TPSA considered alone) to 61%. The cutoff level for TPSA was determined at 4 ng/mL and for the proportion of FPSA at 21.1%. CONCLUSIONS: These data indicate that the differentiation between CaP and BPH can be considerably improved by measuring both FPSA and TPSA and calculating the ratio of the one to the other.