RESUMO
Pharmacogenomically relevant markers of drug response and adverse drug reactions are known to vary in frequency across populations. We examined minor allele frequencies (MAFs), genetic diversity (FST) and population structure of 1156 genetic variants (including 42 clinically actionable variants) in 212 genes involved in drug absorption, distribution, metabolism and excretion (ADME) in 19 populations (n=1478). There was wide population differentiation in these ADME variants, reflected in the range of mean MAF (ΔMAF) and FST. The largest mean ΔMAF was observed in African ancestry populations (0.10) and the smallest mean ΔMAF in East Asian ancestry populations (0.04). MAFs ranged widely, for example, from 0.93 for single-nucleotide polymorphism (SNP) rs9923231, which influences warfarin dosing to 0.01 for SNP rs3918290 associated with capecitabine metabolism. ADME genetic variants show marked variation between and within continental groupings of populations. Enlarging the scope of pharmacogenomics research to include multiple global populations can improve the evidence base for clinical translation to benefit all peoples.
Assuntos
Farmacogenética , Grupos Populacionais , Padrões de Prática Médica , Frequência do Gene , Humanos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Alagille syndrome (AGS) is an autosomal-dominant disorder characterized by intrahepatic cholestasis and abnormalities of heart, eye and vertebrae, as well as a characteristic facial appearance. Identification of rare AGS patients with cytogenetic deletions has allowed mapping of the gene of 20p12. We have generated a cloned contig of the critical region and used fluorescent in situ hybridization on cells from patients with submicroscopic deletions to narrow the candidate region to only 250 kb. Within this region we identified JAG1, the human homologue of rat Jagged1, which encodes a ligand for the Notch receptor. Cell-cell Jagged/Notch interactions are known to be critical for determination of cell fates in early development, making this an attractive candidate gene for a developmental disorder in humans. Determining the complete exon-intron structure of JAG1 allowed detailed mutational analysis of DNA samples from non-deletion AGS patients, revealing three frame-shift mutations, two splice donor mutations and one mutation abolishing RNA expression from the altered allele. We conclude that AGS is caused by haploinsufficiency of JAG1.
Assuntos
Síndrome de Alagille/genética , Proteínas de Membrana/genética , Receptores de Superfície Celular , Fatores de Transcrição , Proteínas de Ligação ao Cálcio , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 20/genética , Clonagem Molecular , Análise Mutacional de DNA , Primers do DNA , Éxons/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Hibridização in Situ Fluorescente , Peptídeos e Proteínas de Sinalização Intercelular , Íntrons/genética , Proteína Jagged-1 , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mutação , Polimorfismo Conformacional de Fita Simples , Splicing de RNA/genética , Receptor Notch1 , Análise de Sequência de DNA , Deleção de Sequência/genética , Proteínas Serrate-JaggedRESUMO
Genetic testing of tumor susceptibility genes is now recommended in most patients with pheochromocytoma or paraganglioma (PPGL), even in the absence of a syndromic presentation. Once a mutation is diagnosed there is rarely follow-up validation to assess the possibility of misdiagnosis. This study prospectively examined the prevalence of von Hippel-Lindau (VHL) gene mutations among 182 patients with non-syndromic PPGLs. Follow-up in positive cases included comparisons of biochemical and tumor gene expression data in 64 established VHL patients, with confirmatory genetic testing in cases with an atypical presentation. VHL mutations were detected by certified laboratory testing in 3 of the 182 patients with non-syndromic PPGLs. Two of the 3 had an unusual presentation of diffuse peritoneal metastases and substantial increases in plasma metanephrine, the metabolite of epinephrine. Tumor gene expression profiles in these 2 patients also differed markedly from those associated with established VHL syndrome. One patient was diagnosed with a partial deletion by Southern blot analysis and the other with a splice site mutation. Quantitative polymerase chain reaction, multiplex ligation-dependent probe amplification, and comparative genomic hybridization failed to confirm the partial deletion indicated by certified laboratory testing. Analysis of tumor DNA in the other patient with a splice site alteration indicated no loss of heterozygosity or second hit point mutation. In conclusion, VHL germline mutations represent a minor cause of non-syndromic PPGLs and misdiagnoses can occur. Caution should therefore be exercised in interpreting positive genetic test results as the cause of disease in patients with non-syndromic PPGLs.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Mutação em Linhagem Germinativa , Feocromocitoma/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Doença de von Hippel-Lindau/genética , Adolescente , Neoplasias das Glândulas Suprarrenais/diagnóstico , Neoplasias das Glândulas Suprarrenais/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Catecolaminas/metabolismo , Criança , Erros de Diagnóstico , Feminino , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Feocromocitoma/diagnóstico , Feocromocitoma/metabolismo , Prevalência , Adulto Jovem , Doença de von Hippel-Lindau/diagnóstico , Doença de von Hippel-Lindau/metabolismoRESUMO
Neurofibromatosis type 1 (NF1) is a common tumor-predisposition disorder due to germline mutations in the tumor suppressor gene NF1. A virtually pathognomonic finding of NF1 is the plexiform neurofibroma (PN), a benign, likely congenital tumor that arises from bi-allelic inactivation of NF1. PN can undergo transformation to a malignant peripheral nerve sheath tumor, an aggressive soft-tissue sarcoma. To better understand the non-NF1 genetic contributions to PN pathogenesis, we performed whole-exome sequencing, RNASeq profiling and genome-wide copy-number determination for 23 low-passage Schwann cell cultures established from surgical PN material with matching germline DNA. All resected tumors were derived from routine debulking surgeries. None of the tumors were considered at risk for malignant transformation at the time; for example, there was no pain or rapid growth. Deep (~500X) NF1 exon sequencing was also conducted on tumor DNA. Non-NF1 somatic mutation verification was performed using the Ampliseq/IonTorrent platform. We identified 100% of the germline NF1 mutations and found somatic NF1 inactivation in 74% of the PN. One individual with three PNs had different NF1 somatic mutations in each tumor. The median number of somatic mutations per sample, including NF1, was one (range 0-8). NF1 was the only gene that was recurrently somatically inactivated in multiple tumors. Gene Set Enrichment Analysis of transcriptome-wide tumor RNA sequencing identified five significant (FDR<0.01) and seven trending (0.01⩽FDR<0.02) gene sets related to DNA replication, telomere maintenance and elongation, cell cycle progression, signal transduction and cell proliferation. We found no recurrent non-NF1 locus copy-number variation in PN. This is the first multi-sample whole-exome and whole-transcriptome sequencing study of NF1-associated PN. Taken together with concurrent copy-number data, our comprehensive genetic analysis reveals the primacy of NF1 loss as the driver of PN tumorigenesis.
Assuntos
Neurofibroma Plexiforme/patologia , Neurofibromatose 1/patologia , Neurofibromina 1/deficiência , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Replicação do DNA , Dosagem de Genes , Genes Supressores de Tumor , Mutação em Linhagem Germinativa , Humanos , Neurofibroma Plexiforme/genética , Neurofibroma Plexiforme/metabolismo , Neurofibromatose 1/genética , Neurofibromatose 1/metabolismo , Neurofibromina 1/genética , TranscriptomaRESUMO
BACKGROUND: The molecular mechanisms underlying the progression of prostate cancer during hormonal therapy have remained poorly understood. In this study, we developed a new strategy for the identification of differentially expressed genes in hormone-refractory human prostate cancer by use of a combination of complementary DNA (cDNA) and tissue microarray technologies. METHODS: Differences in gene expression between hormone-refractory CWR22R prostate cancer xenografts (human prostate cancer transplanted into nude mice) and a xenograft of the parental, hormone-sensitive CWR22 strain were analyzed by use of cDNA microarray technology. To validate the data from cDNA microarrays on clinical prostate cancer specimens, a tissue microarray of specimens from 26 prostates with benign prostatic hyperplasia, 208 primary prostate cancers, and 30 hormone-refractory local recurrences was constructed and used for immunohistochemical detection of protein expression. RESULTS: Among 5184 genes surveyed with cDNA microarray technology, expression of 37 (0.7%) was increased more than twofold in the hormone-refractory CWR22R xenografts compared with the CWR22 xenograft; expression of 135 (2.6%) genes was reduced by more than 50%. The genes encoding insulin-like growth factor-binding protein 2 (IGFBP2) and 27-kd heat-shock protein (HSP27) were among the most consistently overexpressed genes in the CWR22R tumors. Immunohistochemical analysis of tissue microarrays demonstrated high expression of IGFBP2 protein in 100% of the hormone-refractory clinical tumors, in 36% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). Overexpression of HSP27 protein was demonstrated in 31% of the hormone-refractory tumors, in 5% of the primary tumors, and in 0% of the benign prostatic specimens (two-sided P =.0001). CONCLUSIONS: The combination of cDNA and tissue microarray technologies enables rapid identification of genes associated with progression of prostate cancer to the hormone-refractory state and may facilitate analysis of the role of the encoded gene products in the pathogenesis of human prostate cancer.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , DNA Complementar/efeitos dos fármacos , DNA de Neoplasias/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Animais , DNA Complementar/análise , DNA de Neoplasias/análise , Proteínas de Choque Térmico/análise , Proteínas de Choque Térmico/genética , Humanos , Imuno-Histoquímica , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/análise , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/genética , Masculino , Camundongos , Camundongos Nus , Recidiva Local de Neoplasia , Hiperplasia Prostática/genética , Neoplasias da Próstata/química , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Falha de TratamentoRESUMO
We have reported previously that about 15% of anaplastic astrocytomas and glioblastomas show amplification and overexpression of one or more genes from chromosomal segment 12q13-q15 (G. Reifenberger et al., Cancer Res., 54, 4299-4303, 1994). The genes most frequently amplified and overexpressed were CDK4 (with coamplification of SAS) and MDM2. Because individual malignant gliomas showed CDK4/SAS amplification but no MDM2 amplification and vice versa, the possibility remained of a common amplification target gene located between CDK4 and MDM2. We have addressed this question by performing a detailed amplicon mapping of a series of 24 primary malignant gliomas and two glioblastoma cell lines with 12q13-q15 amplification. All tumors and cell lines were analyzed at eight gene loci and six anonymous loci from 12q13-q15, including seven loci located between CDK4 and MDM2. These studies revealed two centers of amplification, one at CDK4/SAS and the other at MDM2. A number of loci located close to either MDM2 or CDK4/SAS, including the genes GADD153, GLI, RAP1B, A2MR, and IFNG, were found to be coamplified in some tumors but not overexpressed consistently. All amplicons were discontinuous between CDK4/SAS and MDM2. Our results thus exclude a common amplification target between CDK4/SAS and MDM2 and provide additional evidence that these genes represent two independent targets of selection.
Assuntos
Neoplasias Encefálicas/genética , Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 12/genética , Quinases Ciclina-Dependentes/genética , Amplificação de Genes/genética , Glioblastoma/genética , Proteínas de Neoplasias/genética , Proteínas Nucleares , Proteínas Proto-Oncogênicas/genética , Adulto , Idoso , Quinase 4 Dependente de Ciclina , Sondas de DNA/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-mdm2RESUMO
The development and use of molecular-based therapy for breast cancer and other human malignancies will require a detailed molecular genetic analysis of patient tissues. The recent development of laser capture microdissection and high density cDNA arrays now provides a unique opportunity to generate gene expression profiles of cells from various stages of tumor progression as it occurs in the actual neoplastic tissue milieu. We report the combined use of laser capture microdissection and high-throughput cDNA microarrays to monitor in vivo gene expression levels in purified normal, invasive, and metastatic breast cell populations from a single patient. These in vivo gene expression profiles were verified by real-time quantitative PCR and immunohistochemistry. The combined use of laser capture microdissection and cDNA microarray analysis provides a powerful new approach to elucidate the in vivo molecular events surrounding the development and progression of breast cancer and is generally applicable to the study of malignancy.
Assuntos
Neoplasias da Mama/genética , Análise Citogenética , DNA Complementar , Expressão Gênica , Proteínas de Neoplasias/genética , Neoplasias da Mama/patologia , Progressão da Doença , Dissecação/métodos , Estudos de Viabilidade , Feminino , Humanos , Imuno-Histoquímica , Lasers , Proteínas de Neoplasias/análise , Reação em Cadeia da Polimerase/métodos , RNA Neoplásico/análiseRESUMO
We hypothesize that elevation of Nm23-H1 expression in micrometastatic breast cancer cells may inhibit their metastatic colonization and further invasion, and induce differentiation, thus resulting in a clinical benefit. The current study investigated the possible contribution of DNA methylation to the regulation of Nm23-H1 expression, based on the observation that two CpG islands are present in its promoter. 5-Aza-2'-deoxycytidine (5-Aza-CdR), a DNA methylation inhibitor, increased the Nm23-H1 expression of 5 of 11 human breast carcinoma cell lines in vitro, including 3 of 3 metastatically competent lines. Increased Nm23-H1 expression was accompanied by a reduction in motility in vitro, with minimal effect on proliferation. Both increased Nm23-H1 expression and decreased motility were observed using low (75 nM) concentrations of 5-Aza-CdR. Array analysis of MDA-MB-231 breast carcinoma cells treated with 5-Aza-CdR confirmed the elevation of nm23-H1 mRNA, whereas relatively few other genes exhibited altered expression. Bisulfite sequencing of the two CpG islands in a panel of cell lines and in 20 infiltrating ductal carcinomas revealed that one island (-3090 bp to -3922 bp) exhibited infrequent differential methylation. The data indicate that DNA methylation inhibitors can directly or indirectly cause both elevation of Nm23-H1 expression and decreased function in one aspect of metastasis, motility.
Assuntos
Azacitidina/análogos & derivados , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Movimento Celular/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica/fisiologia , Genes Supressores de Tumor , Proteínas Monoméricas de Ligação ao GTP/genética , Núcleosídeo-Difosfato Quinase , Fatores de Transcrição/genética , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Ilhas de CpG/genética , Metilação de DNA/efeitos dos fármacos , Decitabina , Humanos , Proteínas Monoméricas de Ligação ao GTP/biossíntese , Nucleosídeo NM23 Difosfato Quinases , Metástase Neoplásica , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
Gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the digestive tract, are believed to arise from the interstitial cells of Cajal. GISTs are characterized by mutations in the proto-oncogene KIT that lead to constitutive activation of its tyrosine kinase activity. The tyrosine kinase inhibitor STI571, active against the BCR-ABL fusion protein in chronic myeloid leukemia, was recently shown to be highly effective in GISTs. We used 13,826-element cDNA microarrays to define the expression patterns of 13 KIT mutation-positive GISTs and compared them with the expression profiles of a group of spindle cell tumors from locations outside the gastrointestinal tract. Our results showed a remarkably distinct and uniform expression profile for all of the GISTs. In particular, hierarchical clustering of a subset of 113 cDNAs placed all of the GIST samples into one branch, with a Pearson correlation >0.91. This homogeneity suggests that the molecular pathogenesis of a GIST results from expansion of a clone that has acquired an activating mutation in KIT without the extreme genetic instability found in the common epithelial cancers. The results provide insight into the histogenesis of GIST and the clinical behavior of this therapeutically responsive tumor.
Assuntos
Neoplasias Gastrointestinais/genética , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Sarcoma/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Aminoácidos , Feminino , Neoplasias Gastrointestinais/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-kit/biossíntese , Sarcoma/metabolismo , Células Estromais/patologiaRESUMO
Drug resistance in cancer is a major obstacle to successful chemotherapy. Cancer cells exposed to antitumor drugs may be directly induced to express a subset of genes that could confer resistance, thus allowing some cells to escape killing and form the relapsed resistant tumor. Alternatively, some cancer cells may be expressing an array of genes that could confer intrinsic resistance, and exposure to cytotoxic drugs select for the survival of these cells that form the relapsed tumor. We have used cDNA microarray to monitor the expression profiles of MCF-7 cells that are either transiently treated with doxorubicin or selected for resistance to doxorubicin. Our results showed that transient treatment with doxorubicin altered the expression of a diverse group of genes in a time-dependent manner. A subset of the induced genes was also found to be constitutively overexpressed in cells selected for resistance to doxorubicin. This distinct set of overlapping genes may represent the signature profile of doxorubicin-induced gene expression and resistance in cancer cells. Our studies demonstrate the feasibility of obtaining potential molecular profile or fingerprint of anticancer drugs in cancer cells by cDNA microarray, which might yield further insights into the mechanisms of drug resistance and suggest alternative methods of treatment.
Assuntos
Antineoplásicos/farmacologia , DNA Complementar/genética , DNA de Neoplasias/genética , Doxorrubicina/farmacologia , Perfilação da Expressão Gênica , Expressão Gênica/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , DNA Complementar/metabolismo , DNA de Neoplasias/metabolismo , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
Androgen deprivation therapy for advanced prostate cancer is often effective, but not curative. Molecular pathways mediating the therapeutic response and those contributing to the subsequent hormone-refractory cell growth remain poorly understood. Here, cDNA microarray analysis of human CWR22 prostate cancer xenografts during the course of androgen deprivation therapy revealed distinct global gene expression profiles in primary, regressing and recurrent tumors. Elucidation of the genes involved in the transition between these states implicated specific molecular mechanisms in therapy failure and tumor progression. First, we identified a set of androgen-responsive genes whose expression decreased during the therapy response, but was then systematically restored in the recurrent tumors. In addition, altered expression of genes that encode known targets of rapamycin or that converge on the PI3K/AKT/FRAP pathway was observed in the recurrent tumors. Further suggestion for the involvement of these genes in hormone-refractory prostate cancer came from the observation that cells established from the recurrent xenografts were strongly inhibited in vitro by rapamycin. The results of this functional genomic analysis suggest that the combined effect of re-expression of androgen-responsive genes as well as the activation of rapamycin-sensitive signaling may drive prostate cancer progression, and contribute to the failure of androgen-deprivation therapy.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/metabolismo , Sirolimo/farmacologia , Algoritmos , Animais , Sobrevivência Celular/efeitos dos fármacos , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Humanos , Masculino , Camundongos , Camundongos Nus , Modelos Biológicos , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/metabolismo , Transdução de Sinais , Software , Fatores de Tempo , Células Tumorais CultivadasRESUMO
We developed a novel integrative genomic tool called GRANITE (Genetic Regulatory Analysis of Networks Investigational Tool Environment) that can effectively analyze large complex data sets to generate interactive networks. GRANITE is an open-source tool and invaluable resource for a variety of genomic fields. Although our analysis is confined to static expression data, GRANITE has the capability of evaluating time-course data and generating interactive networks that may shed light on acute versus chronic treatment, as well as evaluating dose response and providing insight into mechanisms that underlie therapeutic versus sub-therapeutic doses or toxic doses. As a proof-of-concept study, we investigated lithium (Li) response in bipolar disorder (BD). BD is a severe mood disorder marked by cycles of mania and depression. Li is one of the most commonly prescribed and decidedly effective treatments for many patients (responders), although its mode of action is not yet fully understood, nor is it effective in every patient (non-responders). In an in vitro study, we compared vehicle versus chronic Li treatment in patient-derived lymphoblastoid cells (LCLs) (derived from either responders or non-responders) using both microRNA (miRNA) and messenger RNA gene expression profiling. We present both Li responder and non-responder network visualizations created by our GRANITE analysis in BD. We identified by network visualization that the Let-7 family is consistently downregulated by Li in both groups where this miRNA family has been implicated in neurodegeneration, cell survival and synaptic development. We discuss the potential of this analysis for investigating treatment response and even providing clinicians with a tool for predicting treatment response in their patients, as well as for providing the industry with a tool for identifying network nodes as targets for novel drug discovery.
Assuntos
Antimaníacos/uso terapêutico , Transtorno Bipolar/tratamento farmacológico , Compostos de Lítio/uso terapêutico , MicroRNAs/genética , Farmacogenética/métodos , Estatística como Assunto/métodos , Adulto , Feminino , Genômica/instrumentação , Genômica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Farmacogenética/instrumentação , Estatística como Assunto/instrumentação , Resultado do Tratamento , Adulto JovemRESUMO
Alagille syndrome (AGS) is an autosomal dominant, developmental disorder affecting multiple organ systems including liver, heart, vertebrae, eye and face. Recurrent deletions of the 20p12 region led to the localization, and ultimately to the identification of mutations in the Jagged1 gene (JAG1) in AGS patients. A translocation t(3;20)(q13.3;p12.2) in an AGS patient was characterized using fluorescent in situ hybridization (FISH). The involvement of 3q and 20p in this translocation was demonstrated using probes for 3q and 20p. Three overlapping YAC clones, 940D11, 953A2, and 675G11 extending to nearly 4 Mb including the JAG1 were used as probes for FISH analysis to define the translocation breakpoint. The translocated chromosome was found to have a deletion of more than 3 Mb including the entire JAG1 gene. The observation of an accompanying large deletion, revealed by molecular characterization of the t(3;20) translocation, is similar to the only other translocation reported in an AGS patient; a t(2;20) translocation was also found to have a large deletion of the JAG1 region at 20p12.
Assuntos
Síndrome de Alagille/genética , Deleção Cromossômica , Cromossomos Humanos Par 20/genética , Cromossomos Humanos Par 3/genética , Proteínas/genética , Translocação Genética/genética , Síndrome de Alagille/diagnóstico , Proteínas de Ligação ao Cálcio , Humanos , Lactente , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular , Proteína Jagged-1 , Masculino , Proteínas de Membrana , Proteínas Serrate-JaggedRESUMO
Selective COX-2 inhibitors reduce adenoma formation and cancer progression in rodent models of colorectal cancer. To assess the therapeutic activity of selective COX-2 inhibitors, we tested the effect of SC-58125 treatment on the growth of human colon carcinoma cells in nude mice. Delaying treatment by 2, 4, or 7 weeks following implantation of the carcinoma cells resulted in a significant inhibition of tumor growth. Furthermore, short-term (48 hours) treatment with SC-58125 was sufficient to attenuate tumor growth for up to 15 days. SC-58125 treatment did not alter the rate at which cells underwent apoptosis, but did result in a delayed progression through the cell cycle at the G(2)/M transition. Accordingly, p34(cdc2) protein levels and activity were decreased following SC-58125 treatment. We conclude that SC-58125 primarily exerts a cytostatic effect in vivo, which is likely to be mediated through inhibition of progression through the G(2)/M phase of the cell cycle.
Assuntos
Divisão Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Inibidores de Ciclo-Oxigenase/uso terapêutico , Isoenzimas/antagonistas & inibidores , Pirazóis/uso terapêutico , Animais , Apoptose , Divisão Celular/fisiologia , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Immunoblotting , Isoenzimas/genética , Proteínas de Membrana , Camundongos , Camundongos Nus , Índice Mitótico , Prostaglandina-Endoperóxido Sintases/genética , RNA/análise , RNA/metabolismo , Sais de Tetrazólio , Tiazóis , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Pheochromocytomas in von Hippel-Lindau (VHL) syndrome produce exclusively norepinephrine, whereas those in multiple endocrine neoplasia type 2 (MEN 2) produce epinephrine. This study examined the pathways activated in VHL-associated pheochromocytomas by comparing gene expression profiles in VHL and MEN 2 tumors in relationship to profiles in sporadic norepinephrine- and epinephrine-producing tumors. Larger and more distinct differences in gene expression among hereditary than sporadic tumors indicated the importance of the underlying mutation to gene expression profiles. Many of the genes over-expressed in VHL compared with MEN 2 tumors were clearly linked to the hypoxia-driven angiogenic pathways that are activated in VHL-associated tumorigenesis. Such genes included those for the glucose transporter, vascular endothelial growth factor (VEGF), placental growth factor, angiopoietin 2, tie-1, VEGF receptor 2 and its coreceptor, neuropilin-1. Other up-regulated genes, such as connective tissue growth factor, cysteine-rich 61, matrix metalloproteinase 1, vascular endothelial cadherin, tenascin C, stanniocalcin 1, and cyclooxygenases 1 and 2 are known to be involved in VEGF-regulated angiogenesis. Shared differences in expression of subsets of genes in norepinephrine- versus epinephrine-producing hereditary and sporadic pheochromocytomas indicated other differences in gene expression that may underlie the biochemical phenotype. Over-expression of the hypoxia-inducible transcription factor, HIF-2alpha, in norepinephrine-predominant sporadic and VHL tumors compared with epinephrine-producing tumors indicates that expression of this gene depends on the noradrenergic biochemical phenotype. The findings fit with the known expression of HIF-2alpha in norepinephrine-producing cells of the sympathetic nervous system and might explain both the development and noradrenergic biochemical phenotype of pheochromocytomas in VHL syndrome.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Feocromocitoma/genética , Doença de von Hippel-Lindau/genética , Adolescente , Neoplasias das Glândulas Suprarrenais/complicações , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Criança , Epinefrina , Feminino , Perfilação da Expressão Gênica , Humanos , Hipóxia , Masculino , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla Tipo 2a/complicações , Neoplasia Endócrina Múltipla Tipo 2a/genética , Norepinefrina , Análise de Sequência com Séries de Oligonucleotídeos , Feocromocitoma/complicações , Doença de von Hippel-Lindau/complicaçõesRESUMO
We studied the effect of treatment with the Angiotensin II AT(1) receptor antagonist candesartan (0.3 mg/kg/day via osmotic minipumps for 4 weeks compared with administration of vehicle) in brain microvessels in adult spontaneously hypertensive rats (SHR) that were vulnerable to stroke and normotensive control rats (WKY). At the dose administered, candesartan normalized blood pressure in SHR without significantly affecting blood pressure in WKY rats. We performed the gene expression analysis in rat brain microvessels using the Affymetrix Gene Chip Expression Analysis Technique. From a total of 8,799 probe array sets analyzed, we found abundant abnormalities in gene expression in SHR. Because stress has been suggested as a precipitant factor in brain ischemia and treatment with AT(1) receptor antagonist candesartan prevents the hormonal and sympathoadrenal reaction to isolation stress and protects from stress-induced gastric ulcers, we focused on the expression of stress-related genes. We found a higher number of probe array sets modified by candesartan treatment in normotensive WKY rats than in hypertensive SHR. AT(1) receptor blockade decreased the transcription levels of the stress-related tyrosine kinase receptor, stathmin, and fibroblast growth receptor genes in WKY and SHR rats. Our results indicate that Angiotensin II and its AT(1) receptors can influence gene expression independently of the effects on blood pressure. In addition, AT(1) receptor regulation of stress-related genes in brain microvessels may explain the proposed association between stress and ischemic disorders of the brain.
Assuntos
Angiotensina II/metabolismo , Antagonistas de Receptores de Angiotensina , Regulação para Baixo , Estresse Fisiológico/genética , Animais , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Endogâmicos SHR , Ratos Endogâmicos WKY , Receptores de Angiotensina/metabolismoRESUMO
Chromogranin A (CGA) is a major secretory protein present in the soluble matrix of chromaffin granules of neuroendocrine cells and tumours, such as phaeochromocytomas. CGA has several functions, some of which may be involved in the distinct phenotypic differences of phaeochromocytomas in patients with von Hippel-Lindau (VHL) syndrome compared to multiple endocrine neoplasia type 2 (MEN 2). In this study, we therefore compared tumour and plasma levels of CGA in patients with phaeochromocytoma associated with the two syndromes. We show that phaeochromocytomas from MEN 2 patients express substantially more CGA than tumours from VHL patients at both the mRNA (3-fold greater) and protein (20-fold) level. We further show that relative to increases in plasma catecholamines, patients with phaeochromocytomas associated with MEN 2 have higher plasma concentrations of CGA than those with tumours in VHL syndrome. These data supplement other observations that phaeochromocytomas in VHL compared to MEN 2 patients express lower amounts of catecholamines and other chromaffin granule cargo, such as chromogranin B and neuropeptide Y. Possibly the differences in tumour CGA expression may contribute to differences in secretory vesicle formation and secretion in the two types of tumours. Alternatively the differences in expression in CGA and other secretory constituents may reflect downregulation of the entire regulated secretory pathway in VHL compared to MEN 2 tumours.
Assuntos
Neoplasias das Glândulas Suprarrenais/complicações , Neoplasias das Glândulas Suprarrenais/metabolismo , Cromogranina A/metabolismo , Neoplasia Endócrina Múltipla Tipo 2a/complicações , Feocromocitoma/complicações , Feocromocitoma/metabolismo , Doença de von Hippel-Lindau/complicações , Adolescente , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Western Blotting , Catecolaminas/sangue , Criança , Cromogranina A/sangue , Cromogranina A/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2a/patologia , Feocromocitoma/genética , Feocromocitoma/patologia , Reação em Cadeia da Polimerase , Análise de Regressão , Carga Tumoral , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/patologiaRESUMO
The genes encoding enzymes involved in fatty acid metabolism are regulated by sterols. Stearoyl CoA desaturase, a key enzyme in the synthesis of unsaturated fatty acyl-CoAs is transcriptionally regulated by fat-free diet and sterols. To identify other genes that are induced in rat liver by fat-free diet we performed an in vivo gene expression profile analysis using DNA microarrays. Here we report that among the genes highly expressed is emerin, an integral protein of the inner nuclear membrane. Mutated or nonexpressed emerin occurs in patients with muscular dystrophy. Sterol regulatory element binding proteins activate the transcription of several sterol regulated genes. To investigate whether sterol regulatory element binding proteins or indirectly cholesterol activates the transcription of stearoyl CoA desaturase and emerin, we cultured Chinese hamster ovary (CHO), either in cholesterol-rich or cholesterol-depleted mediums. We also transiently transfected the cell culture with plasmid encoding sterol regulatory element binding proteins in cholesterol-rich medium. Our data show that cholesterol-supplemented media as well as the transient transfection induced the expression of stearoyl CoA desaturase RNA 3.5- and 7-fold respectively. However, the RNA level of emerin was not altered under these conditions, implying that the parallel induction of emerin is independent of the sterol regulatory element binding regulation pathway.
Assuntos
Fígado/metabolismo , Proteínas de Membrana/genética , Estearoil-CoA Dessaturase/genética , Timopoietinas/genética , Fatores de Transcrição , Grupos de População Animal , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Células CHO , Colesterol/metabolismo , Cricetinae , Proteínas de Ligação a DNA/genética , Fígado/enzimologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Estearoil-CoA Dessaturase/biossíntese , Proteína de Ligação a Elemento Regulador de Esterol 1 , Timopoietinas/biossíntese , Ativação Transcricional , TransfecçãoRESUMO
It has been increasingly recognized that homogeneously staining regions (hsr) in human cancers may be complex structures composed of large amplified DNA domains containing multiple genes. It is therefore important to devise strategies for the rapid isolation of cDNAs expressed from these structures. Using a procedure we term microdissection mediated cDNA capture, we recovered hsr specific cDNAs from two different human tumors. The glioblastoma cell line TX3868 and the human sarcoma cell line OsA-CL carry hsrs containing amplified sequences from chromosome 12q13-15. We recovered 17 hsr specific cDNAs following microdissection of these hsrs which had been previously hybridized in situ with linkered cDNA. Northern blot analysis with these cDNAs revealed hybridization to distinct transcripts in OsA-CL RNA and TX3868 RNA. None of the OsA-CL cDNA clones showed cross hybridization with the TX3868 cDNAs suggesting that despite their coincident band localization on 12q, the OsA-CL and TX3868 amplification units do not completely overlap. These results significantly increase the number of amplified genes assigned to the 12q13-15 amplicon illustrating both the complexity of hsrs derived from this region and the utility of microdissection mediated cDNA capture to gain rapid access to cDNAs transcribed from amplified genes.
Assuntos
DNA Complementar/análise , DNA de Neoplasias/análise , Amplificação de Genes , Neoplasias/genética , Sequência de Bases , Northern Blotting , Southern Blotting , Eletroforese em Gel de Ágar , Glioblastoma/genética , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Mutagênese Insercional , Hibridização de Ácido Nucleico , Osteossarcoma/genética , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
Notch proteins are a family of closely related transmembrane receptors proven to be instrumental in cell fate decisions. Recently, Notch ligands Delta and Jagged have been identified in Drosophila and rat, respectively. We have isolated the human homolog of the rat Jagged1 gene, JAG1, from a CpG island in a YAC clone covering the Alagille syndrome critical region at chromosome 20p12 (tel-SNAP-D20S186-cen). Alagille syndrome is an autosomal dominant disorder characterized by neonatal jaundice, paucity of intrahepatic bile ducts, and abnormalities of the heart, skeleton, and eyes. The human Jagged1 (JAG1), therefore, appears to be a strong candidate gene for this disease. Here we describe the identification, full-length cDNA cloning, expression patterns, and precise physical location of this gene within the Alagille syndrome critical region.