RESUMO
Using real-time RT-PCR in combination with bioinformatics, we have shown for the first time that the treatment of HCT-116 and HT-29 colon cancer cells with two anti-cancer agents (doxycycline or 3,3'-diindolylmethane) results in profound changes in the intracellular content of several lncRNAs (by up to 100 times). Since many of these RNAs are secreted by tumors into the bloodstream, the obtained results provide a basis for developing more sensitive protocols for serological monitoring of tumor relapse and metastasis, as well as for search of new anti-cancer drugs.
Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , RNA Longo não Codificante/biossíntese , RNA Neoplásico/biossíntese , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Humanos , RNA Longo não Codificante/genética , RNA Neoplásico/genéticaRESUMO
Retinoids are signaling molecules that control a wide variety of cellular processes and possess antitumor activity. This work presents a comprehensive description of changes in the expression of 23 genes that regulate retinoid metabolism and signaling in non-small-cell lung cancer tumors compared to adjacent normal tissues obtained using RT-PCR. Even at early stages of malignant transformation, a significant decrease in ADH1B, ADH3, RDHL, and RALDH1 mRNA levels was observed in 82, 79, 73, and 64% of tumor specimens, respectively, and a considerable increase in AKR1B10 mRNA content was observed in 80% of tumors. Dramatic changes in the levels of these mRNAs can impair the synthesis of all-trans retinoic acid, a key natural regulatory retinoid. Apart from that, it was found that mRNA levels of nuclear retinoid receptor genes RXRγ, RARα, RXRα, and gene RDH11 were significantly decreased in 80, 67, 57, and 66% of tumor specimens, respectively. Thus, neoplastic transformation of lung tissue cells is accompanied with deregulated expression of key genes of retinoid metabolism and function.
Assuntos
3-Hidroxiesteroide Desidrogenases/biossíntese , Álcool Desidrogenase/biossíntese , Aldeído Desidrogenase/biossíntese , Aldeído Redutase/biossíntese , Carcinoma Pulmonar de Células não Pequenas/genética , 3-Hidroxiesteroide Desidrogenases/genética , Álcool Desidrogenase/genética , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Aldeído Redutase/genética , Aldo-Ceto Redutases , Carcinoma Pulmonar de Células não Pequenas/patologia , Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Humanos , RNA Mensageiro/biossíntese , Receptores do Ácido Retinoico/biossíntese , Receptores do Ácido Retinoico/genética , Retinal Desidrogenase , Receptor alfa de Ácido Retinoico , Retinoides/genética , Retinoides/metabolismo , Transdução de Sinais/genética , Tretinoína/metabolismoRESUMO
The review describes the changes observed in long noncoding RNA (lncRNA) content and function at various stages of carcinogenesis, as well as the prospects of lncRNA application in cancer prognosis.
RESUMO
Anew immuno-PCR format is described that is based on detection of membrane protein CDH17 in serum exosomes. Format application allows distinction between sera samples of healthy donors and colon cancer patients. Obtained results open a possibility of serological colon cancer diagnosis in high risk groups.
Assuntos
Biomarcadores Tumorais/sangue , Caderinas/sangue , Neoplasias do Colo/sangue , Neoplasias do Colo/diagnóstico , Exossomos/metabolismo , Reação em Cadeia da Polimerase/métodos , Biomarcadores Tumorais/imunologia , Caderinas/imunologia , Neoplasias do Colo/imunologia , Exossomos/efeitos dos fármacos , Feminino , Humanos , MasculinoRESUMO
This review describes current methods of protein immunoanalysis: radioimmunoanalysis, ELISA, immuno-PCR, electrochemical analysis and chromatin immunoprecipitation, as well as main areas of their application.
Assuntos
Imunoensaio/métodos , Proteínas/análise , Proteínas/imunologia , Imunoprecipitação da Cromatina/métodos , Técnicas Eletroquímicas/métodos , Ensaio de Imunoadsorção Enzimática , Reação em Cadeia da Polimerase/métodos , Radioimunoensaio/métodosRESUMO
This review describes the most popular methods of search for serological markers of tumors that are used in clinical setting, provided with comparison of their efficiency.
Assuntos
Biomarcadores Tumorais , Eletroforese em Gel Bidimensional/métodos , Neoplasias , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biologia Computacional , Humanos , Neoplasias/sangue , Neoplasias/diagnóstico , Neoplasias/genética , Proteômica/métodosRESUMO
It was demonstrated that enteric alpha-defensin 5 is undetectable in five blood serum samples of healthy donors, whereas its processed form is present in two out of five serum samples of colon cancer patients. Obtained results open a possibility of serological diagnosis of colon tumors in high risk cancer patients.
Assuntos
Anticorpos , Neoplasias do Colo , alfa-Defensinas/sangue , Biomarcadores Tumorais/sangue , Neoplasias do Colo/sangue , Neoplasias do Colo/patologia , Humanos , alfa-Defensinas/isolamento & purificaçãoRESUMO
This review summarizes current knowledge on the role of tumor exosomes and microvesicles in progression, metastasis, and angiogenesis of tumors, as well as in suppression of adaptive and innate immunity.
Assuntos
Carcinogênese/genética , Neoplasias/genética , Neovascularização Patológica/genética , Imunidade Adaptativa , Exossomos/genética , Exossomos/metabolismo , Humanos , Imunidade Inata , Metástase Neoplásica , Neoplasias/imunologia , Neoplasias/patologia , Neovascularização Patológica/imunologia , Neovascularização Patológica/patologia , Vesículas Transportadoras/genética , Vesículas Transportadoras/metabolismoRESUMO
A new construct of DNA reporter has been designed for protein quantification by immuno-PCR. It has been shown that amplification efficiency of a reporter that contains a fragment of human adenovirus 2 flanked by homoprimer sequences is much higher vs. standard PCR format based on use of two different primers. Application of a new construct and its homoprimer-based detection opens a way to a significant increase in the immuno-PCR sensitivity and the efficiency of single molecule PCR.
Assuntos
Primers do DNA , DNA Viral/genética , Genes Reporter/genética , Reação em Cadeia da Polimerase/métodos , Adenoviridae , Animais , Humanos , Sensibilidade e EspecificidadeRESUMO
This review summarizes currently available data on enteric alpha defensins structure, their functions in the innate and adaptive immunity systems and the role in development of intestinal illnesses.
Assuntos
Imunidade Adaptativa , Defensinas/imunologia , Imunidade Inata , Inflamação/imunologia , Celulas de Paneth/química , Sequência de Aminoácidos , Animais , Defensinas/química , Defensinas/metabolismo , Humanos , Inflamação/metabolismo , Dados de Sequência Molecular , Celulas de Paneth/imunologia , Celulas de Paneth/microbiologia , Conformação Proteica , Gastropatias/metabolismo , Gastropatias/microbiologiaRESUMO
Comparison of protein expression in intestinal and diffuse stomach tumors by 2D gel electrophoresis led to identification of three proteins (SOD2, S100A6, and TXN), which are overexpressed in tumors as compared to normal controls. It was shown, that overexpression of proteins SOD2 and TXN occurs much more frequently in diffuse tumors than in intestinal ones. A control panel of eleven proteins overexpressed in stomach tumors has been selected based on the data of comparative 2D analysis described in the literature. Bioinformatics search for mRNAs encoding proteins from the control panel in Oncomine database (which contains the results of determination of mRNA transcription level in tumor vs. normal samples) demonstrated the coincidence of proteomic and transcriptomic data for seven out of 11 proteins.
Assuntos
Biomarcadores Tumorais/biossíntese , Proteínas de Ciclo Celular/biossíntese , Proteínas de Neoplasias/biossíntese , Proteômica , Proteínas S100/biossíntese , Neoplasias Gástricas/metabolismo , Superóxido Dismutase/biossíntese , Tiorredoxinas/biossíntese , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/genética , Biologia Computacional , Eletroforese em Gel Bidimensional , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Intestinais/genética , Neoplasias Intestinais/metabolismo , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Proteína A6 Ligante de Cálcio S100 , Proteínas S100/análise , Proteínas S100/genética , Neoplasias Gástricas/genética , Superóxido Dismutase/análise , Superóxido Dismutase/genética , Tiorredoxinas/análise , Tiorredoxinas/genéticaRESUMO
A modified method of proteome comparative analysis based on preliminary removal of cell structural proteins by extraction using salt buffer and subsequent separation of extracts by two-dimensional gel electrophoresis was developed. Identification of differentially expressed proteins by mass spectrometry has revealed three proteins with noticeably increased level of synthesis in most samples of papillary thyroid tumors compared to normal tissues. An increase in ubiquitin content was found for the first time. Oncomarker search efficiencies by two-dimensional gel electrophoresis and bioinformatic search were compared.
Assuntos
Proteoma/metabolismo , Proteômica/métodos , Neoplasias da Glândula Tireoide/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma , Carcinoma Papilar , Eletroforese em Gel Bidimensional , Humanos , Espectrometria de Massas , Câncer Papilífero da TireoideRESUMO
LRP1B is a novel candidate tumor suppressor gene that is inactivated by genetic and transcript alterations in nearly 50% of non-small-cell lung cancer cell lines. The gene-encoded protein is highly homologous to the gigantic lipoprotein receptor-related protein 1 (LRP1) that belongs to the family of low-density lipoprotein receptors. Using a combination of PCR-based genome walking and long-distance interexon PCR, we have determined the genomic organization of LRP1B and built a contiguous array of BAC clones spanning this gene. A total of 91 exons, varying in size from 77 bases (exon 87) to 1899 bases (exon 91), were identified in the more than 500-kb-long gene sequence. Comparative analysis of the genomic structures of LRP1B and the homologous LRP1 gene revealed a striking similarity in the location and sizes of their exons.
Assuntos
Genes Supressores de Tumor , Receptores Imunológicos/genética , Cromossomos Artificiais Bacterianos , Éxons , Humanos , Íntrons , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
The family of tumor necrosis factor related apoptosis inducing ligand (TRAIL) receptors, including the pro-apoptotic DR4 and p53-regulated KILLER/DR5, as well as the decoys TRID and TRUNDD, are all located on human chromosome 8p21-22. This region of the genome is frequently altered in head and neck cancer. We previously reported that KILLER/DR5 can be mutationally inactivated in head and neck cancer. Here, we report that the FaDu nasopharyngeal cancer cell line contains an abnormal chromosome 8p21-22 region. In addition, there appears to be a homozygous deletion involving DR4 but not KILLER/DR5 in FaDu cells. The homozygous loss within the DR4 gene encompasses its death domain, which is required for apoptotic signaling. The deletion of DR4 in FaDu cells is associated with resistance to the cytotoxic effects of TRAIL. Re-introduction of wild-type DR4 leads to apoptosis and restores TRAIL sensitivity of FaDu cells. These observations suggest that the death inducing DR4 receptor gene may be a rare target for inactivation in human cancer and that DR4 loss may contribute to resistance to TRAIL therapy.
Assuntos
Deleção de Genes , Neoplasias Nasofaríngeas/genética , Receptores do Fator de Necrose Tumoral/genética , Apoptose , Cromossomos Humanos Par 8 , Humanos , Perda de Heterozigosidade , Repetições de Microssatélites , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Células Tumorais CultivadasRESUMO
A variety of studies suggest that allelic losses at chromosome 2q are associated with aggressive behavior of various forms of human neoplasia. Using a probe to detect homozygous deletions on chromosome 2q21.2 in kidney and bladder cancer cell lines, we identified a new candidate tumor suppressor gene, lipoprotein receptor-related protein-deleted in tumors (LRP-DIT). The predicted LRP-DIT product of 4599 amino acids has extensive homology to a gigantic receptor, LRP1, which mediates endocytosis of multiple proteins from the cell surface. Homozygous deletions in LRP-DIT were detected in 17% (4 of 23) of non-small cell lung cancer (NSCLC) cell lines. The expression of only abnormal transcripts missing portions of the LRP-DIT sequence was demonstrated in an additional 30% (11 of 36) of NSCLC lines. Finally, a missense mutation at codon 3157 was detected in one of four NSCLC lines tested for the large open reading frame. In contrast, no LRP-DIT alterations were identified in a major fraction of SCLC cell lines, indicating that this gene is preferentially inactivated in one histological type of lung cancer. Our data suggest that inactivation of LRP-DIT occurs in at least 40% of NSCLC lines and thus may play an important role in tumorigenesis of NSCLCs.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Cromossomos Humanos Par 2 , Deleção de Genes , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Mutação de Sentido Incorreto , Receptores Imunológicos/genética , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Códon , Endocitose , Marcadores Genéticos , Homozigoto , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Dados de Sequência Molecular , Fases de Leitura Aberta , Células Tumorais CultivadasRESUMO
Representational difference analysis (RDA) was used to generate Y-specific probes by enriching for and cloning the differences between the male (XY) and the female (XX) C57BL/6J mouse genomes. Characterization of 35 clones revealed 12 families related by sequence similarity. One clone from each family was chosen for detailed analysis by Southern blot hybridization, polymerase chain reaction (PCR) on normal and aberrant genomes (Sxr), and fluorescence in situ hybridization. From one difference product we have characterized 12 Y-specific probes for hybridization, created seven male-specific PCR assays, mapped all repeat families, and identified one repeat with a distinct XY homology. We report the first cloning of a Y-specific long interspersed repeat element (LINE) fragment. In total, RDA has identified six novel Y Chromosome repeat families and allowed us to extend the characterization of six known Y repeats. We conclude that this novel use of RDA for whole chromosome subtraction successfully enriches chromosome-specific sequences and is suitable for the rapid generation of new Y Chromosome-specific probes.
Assuntos
Sondas de DNA , Cromossomo Y , Animais , Sequência de Bases , Southern Blotting , Feminino , Hibridização in Situ Fluorescente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
A variety of studies suggests that tumor suppressor loci on chromosome 3p are important in various forms of human neoplasia. Recently, a chromosome 3p14.2 gene called FHIT was discovered and proposed as a candidate tumor suppressor gene in colorectal and other cancers. We evaluated the FHIT gene in a panel of colorectal cancer cell lines and xenografts, which allowed a comprehensive mutational analysis. A transcript containing the complete coding sequence was found to be expressed at robust levels in 29 of 31 cancers tested. The complete sequence of the coding region of the gene was determined and found to be normal in all 29 of these cases. These studies suggest either that FHIT is inactivated by an unusual mechanism or that it plays a role in relatively few colorectal tumors.
Assuntos
Hidrolases Anidrido Ácido , Neoplasias Colorretais/genética , Genes Supressores de Tumor , Proteínas de Neoplasias/genética , Proteínas/genética , Alelos , Sequência de Bases , Cromossomos Humanos Par 3 , DNA de Neoplasias/genética , Estudos de Avaliação como Assunto , Deleção de Genes , Homozigoto , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Representational difference analysis (RDA) is an efficient method for finding the differences between complex genomes. The numerous applications of RDA include the cloning of probes for the detection of genetic lesions in cancer, the identification of sequences from the genomes of unknown pathogens and the rapid isolation of polymorphic markers linked to a trait without the use of pre-existing genetic maps.
Assuntos
Técnicas Genéticas , Genoma , AnimaisRESUMO
We demonstrate the use of representational difference analysis for cloning probes that detect DNA loss and amplification in tumors. Using DNA isolated from human tumor cell lines to drive hybridization against matched normal DNA, we were able to identify six genomic regions that are homozygously deleted in cultured cancer cells. When this method was applied in the reverse way, using normal DNA to drive hybridization against tumor cell DNA, we readily isolated probes detecting amplification. Representational difference analysis was also performed on DNAs derived from tumor biopsies, and we thereby discovered a probe detecting very frequent homozygous loss in colon cancer cell lines and located on chromosome 3p.