RESUMO
L-asparaginase has been used for more than three decades in acute lymphoblastic leukemia (ALL) patients and remains an essential drug in the treatment of ALL. Poor response to L-asparaginase is associated with increased risk of therapeutic failure in ALL. However, both the metabolic perturbation and molecular context of L-asparaginase-treated ALL cells has not been fully elucidated. Here we identify that treatment with L-asparaginase results in metabolic shutdown via the reduction of both glycolysis and oxidative phosphorylation, accompanied by mitochondrial damage and activation of autophagy. The autophagy is involved in reducing reactive oxygen species (ROS) level by eliminating injured mitochondria. Inhibition of autophagy enhances L-asparaginase-induced cytotoxicity and overcomes the acquired resistance to L-asparaginase in ALL cells. The ROS-p53-positive feedback loop is an essential mechanism of this synergistic cytotoxicity. Thus, our findings provide the rationale for the future development of combined treatment of L-asparaginase and anti-autophagy drug in ALL patients.
Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Autofagia/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Estresse Fisiológico/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Imunofluorescência , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Metastasis is a critical factor contributing to poor prognosis in cancer, but the underlying mechanisms of metastasis are still poorly understood. We established a highly metastatic cell subline (HOC313-LM) derived from an oral squamous cell carcinoma cell line (HOC313) for uncovering the mechanisms of metastasis, and identified deoxyhypusine synthase (DHPS) as a metastasis-associated gene within the specific amplification at 19p13.2-p13.13 in HOC313-LM. DHPS-mediated hypusine-modification of eukaryotic translation factor 5A facilitated the translation of RhoA, resulting in the activation of the RhoA signaling pathway and leading to not only increased cell motility, invasion and metastasis of cancer cells in vitro, but also increased tumor growth in vivo. Moreover, the use of N1-Guanyl-1,7-diaminoheptane, a DHPS inhibitor, resulted in a significant decrease in tumor formation in vivo. In patients with esophageal squamous cell carcinoma (ESCC), overexpression of DHPS in ESCC tumors was significantly associated with worse recurrence-free survival, and correlated with distant metastasis. The elucidation of these molecular mechanisms within the hypusine cascade suggests opportunities for novel therapeutic targets in SCC.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias Esofágicas/tratamento farmacológico , Lisina/análogos & derivados , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Proteína rhoA de Ligação ao GTP/genética , Adulto , Idoso , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/efeitos dos fármacos , Diaminas/administração & dosagem , Progressão da Doença , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lisina/biossíntese , Lisina/genética , Masculino , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: SET and MYND domain-containing protein 2 (SMYD2) is a lysine methyltransferase for histone H3, p53 and Rb and inhibits their transactivation activities. In this study, we tested whether SMYD2 (1q42) acts as a cancer-promoting factor by being overexpressed in gastric cancer. METHODS: We analysed 7 gastric cancer cell lines and 147 primary tumor samples of gastric cancer, which were curatively resected in our hospital. RESULTS: SET and MYND domain-containing protein 2 was detected in these cell lines (five out of seven cell lines; 71.4%) and primary tumor samples (fifty-six out of one hundred and forty-seven cases; 38.1%). Knockdown of SMYD2 using specific small interfering RNA inhibited proliferation, migration and invasion of SMYD2-overexpressing cells in a TP53 mutation-independent manner. Overexpression of SMYD2 protein correlated with larger tumor size, more aggressive lymphatic invasion, deeper tumor invasion and higher rates of lymph node metastasis and recurrence. Patients with SMYD2-overexpressing tumours had a worse overall rate of survival than those with non-expressing tumours (P=0.0073, log-rank test) in an intensity and proportion score-dependent manner. Moreover, multivariate analysis demonstrated that SMYD2 was independently associated with worse outcome (P=0.0021, hazard ratio 4.25 (1.69-10.7)). CONCLUSIONS: These findings suggest that SMYD2 has a crucial role in tumor cell proliferation by its overexpression and highlight its usefulness as a prognostic factor and potential therapeutic target in gastric cancer.
Assuntos
Expressão Gênica , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias Gástricas/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Histona-Lisina N-Metiltransferase/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Modelos de Riscos Proporcionais , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/cirurgia , Resultado do Tratamento , Proteína Supressora de Tumor p53/genéticaRESUMO
Chromosomal abnormalities are good guideposts when hunting for cancer-related genes. We analyzed copy number alterations of 163 primary gastric cancers using array-based comparative genomic hybridization and simultaneously performed a genome-wide integrated analysis of copy number and gene expression using microarray data for 58 tumors. We showed that chromosome 6p21 amplification frequently occurred secondary to ERBB2 amplification, was associated with poorer prognosis and caused overexpression of half of the genes mapped. A comprehensive small interfering RNA knockdown of 58 genes overexpressed in tumors identified 32 genes that reduced gastric cancer cell growth. Enforced expression of 16 of these genes promoted cell growth in vitro, and six genes showing more than two-fold activity conferred tumor-forming ability in vivo. Among these six candidates, GLO1, encoding a detoxifying enzyme glyoxalase I (GLO1), exhibited the strongest tumor-forming activity. Coexpression of other genes with GLO1 enhanced growth-stimulating activity. A GLO1 inhibitor, S-p-bromobenzyl glutathione cyclopentyl diester, inhibited the growth of two-thirds of 24 gastric cancer cell lines examined. The efficacy was found to be associated with the mRNA expression ratio of GLO1 to GLO2, encoding glyoxalase II (GLO2), another constituent of the glyoxalase system. GLO1 downregulation affected cell growth through inactivating central carbon metabolism and reduced the transcriptional activities of nuclear factor kappa B and activator protein-1. Our study demonstrates that GLO1 is a novel metabolic oncogene of the 6p21 amplicon, which promotes tumor growth and aberrant transcriptional signals via regulating cellular metabolic activities for energy production and could be a potential therapeutic target in gastric cancer.
Assuntos
Cromossomos Humanos Par 6/genética , Genômica/métodos , Lactoilglutationa Liase/genética , Receptor ErbB-2/genética , Neoplasias Gástricas/genética , Animais , Linhagem Celular Tumoral , Hibridização Genômica Comparativa , Amplificação de Genes , Dosagem de Genes , Glutationa/análogos & derivados , Glutationa/metabolismo , Células HEK293 , Humanos , Lactoilglutationa Liase/metabolismo , Camundongos , NF-kappa B/genética , Células NIH 3T3 , Transdução de Sinais , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Fator de Transcrição AP-1/genéticaRESUMO
Epithelial-mesenchymal transition (EMT) has a major role in cancer progression, as well as normal organ development and human pathology such as organ fibrosis and wound healing. Here, we performed a gene expression array specialized in EMT of colorectal cancer (CRC). From a comprehensive gene expression analysis using epithelial- and mesenchymal-like CRC cell lines, and following the ontology (GO) analysis, SIX1 gene was identified to be an EMT-related gene in CRC. Using SW480 cells stably transfected with a SIX1 expression construct and their control counterparts, we demonstrated that SIX1 overexpression represses CDH1 expression and promotes EMT in CRC. SIX1-induced CDH1 repression and EMT in CRC cells were correlated at least in part with posttranscriptional ZEB1 activation and miR-200-family transcriptional repression. In primary tumors of CRC, in accord with the functional findings, aberrant expression of SIX1 in cancer cells was observed at the disruption of the basement membrane and at the tumor invasive front, where tumor cells underwent EMT in vivo. Taken together, SIX1 overexpression is suggested to occur in carcinogenesis, and contribute to repression of CDH1 expression and promotion of EMT partly through repression of miR-200-family expression and activation of ZEB1 in CRC.
Assuntos
Neoplasias Colorretais/metabolismo , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/metabolismo , Antígenos CD , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Proteínas de Homeodomínio/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Fatores de Transcrição/genética , Transcrição Gênica , Transcriptoma , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Microtubule-associated protein 1 light chain 3 has an important role in autophagy. The human LC3 gene family has five members, LC3A (variant-1: v1 and -2: v2), LC3B, LC3B2 and LC3C. Although a form of LC3B modified by phosphatidylethanolamine (form-II) is localized in autophagosomes, it is not clear whether other LC3 proteins also function in autophagy. Here, we examined the association between autophagy and human LC3 proteins during starvation- or p53-induced autophagy in Saos-2 cells. In an analysis of the intracellular distribution of each LC3 protein fused with GFP, GFP-LC3Av1 was frequently localized in autophagosomes with a punctate pattern, similar to GFP-LC3B. Further, endogenous LC3Av1 generated form-II and mostly localized in LC3B-positive autophagosomes during the induced autophagy. Interestingly, LC3Av1, not LC3B, was frequently inactivated at the transcriptional level in various human cancer cell lines (111/244 cell lines, 45.5%) and its inactivation was due to aberrant DNA methylation in esophageal squamous cell carcinoma (ESCC) cell lines and primary tumors. Restoration of LC3Av1 expression in KYSE170 cells, an LC3Av1-inactivated ESCC cell line, showed the inhibition of tumor growth in vivo. These results suggest that LC3Av1, not only LC3B, functions in autophagy and further, LC3Av1 may be crucial in carcinogenesis.
Assuntos
Autofagia/genética , Proteínas Associadas aos Microtúbulos/genética , Linhagem Celular , Linhagem Celular Tumoral , Metilação de DNA , HumanosRESUMO
The epithelial-mesenchymal transition (EMT) has a crucial role in normal and disease processes including tumor progression. In this study, we first classified epithelial-like and mesenchymal-like oral squamous cell carcinoma (OSCC) cell lines based on expression profiles of typical EMT-related genes using a panel of 18 OSCC cell lines. Then, we performed methylation-based and expression-based analyses of components of the Wnt signaling pathway, and identified WNT7A and WNT10A as genes silenced by mesenchymal-specific DNA hypermethylation in OSCCs. A significant association was revealed between some clinicopathological findings and the DNA methylation status of WNT7A (normal vs tumor, P=0.007; T1-2 vs T3-4, P=0.040; I-III vs IV, P=0.016) and WNT10A (N0-N1 vs N2-N3, P=0.046) in the advanced stages of OSCC. Moreover, we found that E-cadherin expression in cancer cells may be positively regulated by WNT7A, whose expression is negatively regulated by mesenchymal-specific DNA hypermethylation or ZEB1 in mesenchymal-like OSCC cells. Our findings indicate that epithelial-specific gene silencing through mesenchymal-specific DNA hypermethylation may stabilize the phenotypic plasticity of cancer cells during EMT/MET.
Assuntos
Carcinoma de Células Escamosas/genética , Metilação de DNA , Transição Epitelial-Mesenquimal , Neoplasias Bucais/genética , Via de Sinalização Wnt , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Proteínas Wnt/genéticaRESUMO
Mutations of the CASK gene are associated with X-linked mental retardation with microcephaly and disproportionate brain stem and cerebellar hypoplasia in females. The areas of the cerebrum, corpus callosum, pons, midbrain, and cerebellar vermis and hemisphere and a ratio of cerebrum/corpus callosum areas were measured in 5 female patients with CASK mutations, 67 female controls, and 5 patients with pontine hypoplasia. MR imaging in patients with CASK mutations revealed a normal size of the corpus callosum and a low ratio of the cerebrum/corpus callosum with a reduced area of the cerebrum, pons, midbrain, and cerebellar vermis and hemispheres. The 5 patients with pontine hypoplasia showed thinning of the corpus callosum and a high ratio of the cerebrum/corpus callosum, irrespective of the size of the cerebrum. The normal size of the corpus callosum, which gives an impression of callosal thickening at first glance, may be an imaging clue to detect patients with CASK mutations.
Assuntos
Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Encéfalo/anormalidades , Encéfalo/patologia , Guanilato Quinases/genética , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Imageamento por Ressonância Magnética , Pré-Escolar , Feminino , Predisposição Genética para Doença/genética , Humanos , Lactente , Masculino , Mutação/genéticaRESUMO
Lung cancer shows diverse histological subtypes. Large-cell neuroendocrine cell carcinoma and small-cell lung carcinoma show similar histological features and clinical behaviors, and can be classified as high-grade neuroendocrine carcinoma (HGNEC) of the lung. Here we elucidated the molecular classification of pulmonary endocrine tumors by copy-number profiling. We compared alterations of copy number with the clinical outcome of HGNEC and identified a chromosomal gain of the DEK oncogene locus (6p22.3) that was significantly associated with poor prognosis. We further confirmed that DEK overexpression was associated with poor prognosis in a larger set of HGNEC. Downregulation of DEK by small hairpin RNA led to a marked reduction of in vitro colony formation, in vivo tumorigenicity and chemo-resistance, and was associated with loss of lung cancer stem cell markers. Gene expression profiling revealed that DEK downregulation was associated with altered expression of transcriptional regulators, which specifically include known targets of interchromosomal translocations in hematopoietic tumors, and knockdown of these epigenetic modifiers affected colony formation activity. Our study showed that DEK overexpression, partly through an increase in its gene dose, mediates the activity of global transcriptional regulators and is associated with tumor initiation activity and poor prognosis in HGNEC.
Assuntos
Carcinoma Neuroendócrino/genética , Proteínas Cromossômicas não Histona/genética , Neoplasias Pulmonares/genética , Proteínas Oncogênicas/genética , Carcinoma Neuroendócrino/metabolismo , Carcinoma Neuroendócrino/patologia , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Processos de Crescimento Celular/genética , Movimento Celular/genética , Proteínas Cromossômicas não Histona/biossíntese , Análise por Conglomerados , Regulação para Baixo , Dosagem de Genes , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/patologia , Proteínas Oncogênicas/biossíntese , Proteínas de Ligação a Poli-ADP-Ribose , Prognóstico , RNA Interferente Pequeno/genética , Transcrição GênicaRESUMO
Cellular inhibitor-of-apoptosis protein 1 (cIAP-1) is a member of the inhibitor-of- apoptosis protein family, which predominantly regulates apoptosis. It has been suggested that expression of cIAP-1 correlates with certain clinicopathological features. The possible significance of cIAP-1 expression in cervical lymph node metastasis of tongue squamous cell carcinoma (SCC) is investigated. Seventy-five tongue SCCs were analyzed by immunohistochemistry. cIAP-1 immunoreactivity patterns were nuclear in 38 (51%), cytoplasmic in 47 (63%), and concurrent in 37 (49%) cases. Nuclear, cytoplasmic and concurrent cIAP-1 immunoreactions were significantly correlated with lymph node metastasis in tongue SCCs (P=0.0011, 0.0012, and 0.0006, respectively). The cleaved caspase-3, which is a marker of tumor apoptosis, and Ki-67 index, which is a marker of tumor proliferation, were immunohistochemically examined in 21 tongue SCCs with concurrent nuclear and cytoplasmic cIAP-1 expression and with metastasis, and in 23 tongue SCCs without concurrent nuclear and cytoplasmic cIAP-1 expression and without metastasis. Concurrent cIAP-1 expression was inversely correlated with caspase-3 (P=0.0066), but was positively correlated with Ki-67 expression (P=0.0028). The mode of invasion was associated with lymph node metastasis (P=0.014) and differentiation (P=0.013), but was not correlated with cIAP-1 expression. There was no statistically significant correlation between nuclear or cytoplasmic cIAP-1 expression and the clinicopathological factors of gender, age, clinical stage or differentiation. These results suggest that both patterns of cIAP-1 are useful markers for predicting cervical lymph node metastasis in tongue SCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/patologia , Proteínas Inibidoras de Apoptose/metabolismo , Metástase Linfática/patologia , Neoplasias da Língua/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Escamosas/metabolismo , Caspase 3/metabolismo , Humanos , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Masculino , Pessoa de Meia-Idade , Distribuição Tecidual , Neoplasias da Língua/metabolismo , Adulto JovemRESUMO
BACKGROUND: Patterns of cancer recurrence hold the key to prognosis after curative resection. This retrospective study aimed to identify a predictor and therapeutic candidate for aggressive recurrence of hepatocellular carcinoma (HCC). METHODS: Primary HCC tissues from 107 patients who had curative resection were analysed. Genome-wide gene expression profiles were investigated using a microarray technique, and clustering analysis was carried out based on the first diagnosis of recurrence according to the Milan criteria. Immunohistochemical expression and array-based comparative genomic hybridization (array-CGH) were also assessed. RESULTS: Microarray analysis revealed overexpression of Aurora kinase B, a chromosome passenger protein kinase, as the most significant predictor of the aggressive recurrence of HCC. Aurora kinase B protein expression was significantly associated with aggressive recurrence (P < 0.001) and prognosis (P < 0.001). Multivariable analysis identified Aurora kinase B as the only independent predictor of aggressive recurrence of HCC (P = 0.031). Array-CGH analysis showed that genomic instability was closely related to Aurora kinase B expression (P = 0.011). CONCLUSION: Aurora kinase B is an effective predictor of aggressive HCC recurrence, in relation to the genomic instability. It might be worth considering as a molecular target for the adjuvant therapy of HCC.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/cirurgia , Hepatectomia/mortalidade , Neoplasias Hepáticas/cirurgia , Recidiva Local de Neoplasia/diagnóstico , Proteínas Serina-Treonina Quinases/metabolismo , Aurora Quinase B , Aurora Quinases , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/mortalidade , Intervalo Livre de Doença , Expressão Gênica , Instabilidade Genômica , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/mortalidade , Análise de Sequência com Séries de Oligonucleotídeos , Prognóstico , Proteínas Serina-Treonina Quinases/genética , RNA Neoplásico/análise , Estudos RetrospectivosRESUMO
Multiple myeloma (MM), a progressive hematological neoplasm, is thought to result from multiple genetic events affecting the terminal plasma cell. However, genetic aberrations related to MM are seldom reported. Using our in-house array-based comparative genomic hybridization system to locate candidate target genes with following their expression analysis, we identified POU2AF1 at 11q23.1 as a probable amplification target in MM cell lines. POU2AF1 is a B-cell-specific transcriptional co-activator, which interacts with octamer-binding transcription factors Oct-1 and Oct-2, and augments their function. Downregulation of POU2AF1 expression by specific small-interfering RNA (siRNA) inhibited MM cell growth, whereas ectopic expression of POU2AF1 promoted growth of MM cells. Among putative transcriptional targets for POU2AF1, B-cell maturation factor, TNFRSF17, enhanced its transcription by POU2AF1, and POU2AF1 directly bound to an octamer site within the 5' region of TNFRSF17. Expression level of TNFRSF17 was closely correlated with that of POU2AF1 in cell lines and primary samples of MM, and decreasing TNFRSF17 expression by means of TNFRSF17 siRNA inhibited MM cell growth. Taken together, our results suggest that POU2AF1, when activated by amplification or other mechanisms, may contribute to progression of MM by accelerating growth of MM cells through direct transactivation of one of its target genes, TNFRSF17.
Assuntos
Antígeno de Maturação de Linfócitos B/biossíntese , Linfócitos B/metabolismo , Proliferação de Células , Cromossomos Humanos Par 11/genética , Amplificação de Genes/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Mieloma Múltiplo/patologia , Transativadores/fisiologia , Antígeno de Maturação de Linfócitos B/genética , Antígeno de Maturação de Linfócitos B/fisiologia , Linfócitos B/citologia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Dosagem de Genes , Marcação de Genes , Humanos , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Transativadores/antagonistas & inibidores , Transativadores/genética , Transativadores/metabolismo , Células Tumorais CultivadasRESUMO
The purpose of this study was to investigate the accumulation of genetic alterations during metachronous and/or synchronous development of multifocal low-grade superficial urothelial tumours in the same patient, by using array-based comparative genomic hybridisation (array-CGH) and FGFR mutation analysis. We analysed 24 tumours (pTa-1 G1-2) from five patients. We had previously identified a clonal relationship among the tumours of each patient by microsatellite analysis. This time, unsupervised hierarchical cluster analysis revealed that the tumours from each patient were clustered together independently of the tumours from the other patients. All of the tumours from a single patient showed a set of 2-7 identical regional or whole-arm chromosomal changes. In addition, several individual alterations were also found. Cladistic diagrams revealed that the accumulation of genetic alterations could not be explained by a linear model, and the existence of a hypothetical precursor cell was assumed in four patients. In some cases, FGFR mutation seemed to occur later during multifocal tumour development. Taken together, these findings suggest that low-grade superficial urothelial tumours accumulate minor genetic alterations during multifocal development, although these tumours are genetically stable.
Assuntos
Mutagênese , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Neoplasias Urológicas/genética , Urotélio , Cromossomos Humanos/genética , Análise Mutacional de DNA , Progressão da Doença , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Urológicas/patologia , Urotélio/metabolismo , Urotélio/patologiaRESUMO
Array-based comparative genomic hybridization (array-CGH) has good potential for the high-throughput identification of genetic aberrations in cell genomes. In the course of a program to screen a panel of oral squamous-cell carcinoma (OSCC), cell lines for genomic copy-number aberrations by array-CGH using our in-house arrays, we identified a 3-Mb homozygous deletion at 10p12 in 1 of 18 cell lines (5.6%). Among seven genes located within this region, expression of PRTFDC1 mRNA was not detected in 50% (9/18) or decreased in 5.6% (1/18) of OSCC cell lines, but detected in normal oral epithelia and restored in gene-silenced OSCC cells without its homozygous loss after treatment with 5-aza-2'-deoxycytidine. Among 17 cell lines without a homozygous deletion, the hypermethylation of the PRTFDC1 CpG island, which showed promoter activity, was observed in all nine cell lines with no or reduced PRTFDC1 expression (52.9%). Methylation of this CpG island was also observed in primary OSCC tissues (8/47, 17.0%). In addition, restoration of PRTFDC1 in OSCC cells lacking its expression inhibited cell growth in colony-formation assays, whereas knockdown of PRTFDC1 expression in OSCC cells expressing the gene promoted cell growth. These results suggest that epigenetic silencing of PRTFDC1 by hypermethylation of the CpG island leads to a loss of PRTFDC1 function, which might be involved in squamous cell oral carcinogenesis.
Assuntos
Carcinoma de Células Escamosas/genética , Metilação de DNA , Inativação Gênica , Genes Supressores de Tumor , Neoplasias Bucais/genética , Regiões Promotoras Genéticas , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Cromossomos Humanos Par 10 , Ilhas de CpG , Decitabina , Humanos , Neoplasias Bucais/patologia , Hibridização de Ácido Nucleico , RNA Mensageiro/análiseRESUMO
We previously identified a cluster of prostanoid receptor genes, prostaglandin D2 receptor (PTGDR) and prostaglandin E receptor 2 (PTGER2), as possible targets for DNA methylation in advanced types of neuroblastoma (NB) using bacterial artificial chromosome array-based methylated CpG island amplification method. Among them, in this study, we found that PTGER2 was frequently silenced in NB cell lines, especially in those with MYCN amplification, through epigenetic mechanisms. In NB cell lines, DNA methylation pattern within a part of CpG island was inversely correlated with PTGER2 expression, and histone H3 and H4 deacetylation and histone H3 lysine 9 methylation within the putative promoter region were more directly correlated with silencing of this gene. Methylation of PTGER2 was observed more frequently in advanced-type of primary NBs compared with early-stage tumors. Growth of NB cells lacking endogenous PTGER2 expression was inhibited by restoration of the gene product by transient and stable transfection. A PTGER2-selective agonist, butaprost, increased intracellular cyclic adenosine monophosphate (cAMP) level, inhibited cell growth and induced apoptosis of NB cells stably expressing exogenous PTGER2. 8-Bromo-cAMP also inhibited growth of NB cells lacking PTGER2 expression, but not cells expressing this gene. Taken together, it is suggested that NB cells may lose responsiveness to PTGER2-mediated growth inhibition/apoptosis through epigenetic silencing of PTGER2 and/or disruption of downstream cAMP-dependent pathway during the neuroblastomagenesis.
Assuntos
Genes Supressores de Tumor , Neuroblastoma/genética , Neuroblastoma/patologia , Receptores de Prostaglandina E/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Alprostadil/análogos & derivados , Alprostadil/farmacologia , Processos de Crescimento Celular/efeitos dos fármacos , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ilhas de CpG , Metilação de DNA , Dinoprostona/farmacologia , Progressão da Doença , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Neuroblastoma/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores Imunológicos/biossíntese , Receptores Imunológicos/genética , Receptores de Prostaglandina/biossíntese , Receptores de Prostaglandina/genética , Receptores de Prostaglandina E/biossíntese , Receptores de Prostaglandina E Subtipo EP2RESUMO
Epigenetic alterations and the resulting inactivation of tumor suppressor genes often contribute to the development of various cancers. To identify novel candidates that may be silenced by aberrant methylation in esophageal squamous-cell carcinoma (ESCC), we analysed ESCC cell lines by a recently developed method known as bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA), and selected candidates through BAMCA-assisted strategy. In the course of this program, we identified frequent CpG methylation-dependent silencing of the gene encoding cellular retinoic acid binding protein 1 (CRABP1) in our panel of ESCC cell lines. Expression of CRABP1 mRNA was restored in gene-silenced ESCC cells after treatment with 5-aza 2'-deoxycytidine. The DNA methylation status of the CRABP1 CpG island with clear promoter activity correlated inversely with expression of this gene. CpG methylation of CRABP1 was frequently observed in primary ESCC tissues as well. Restoration of CRABP1 expression in ESCC cells lacking the protein reduced cell growth by inducing arrest at G(0)-G(1), whereas knockdown of the gene in cells expressing CRABP1 promoted cell growth. Among 113 primary ESCC tumors, the absence of immunoreactive CRABP1 was significantly associated with de-differentiation of cancer cells and with distant lymph-node metastases in the patients. These results indicate that CRABP1 appears to have a tumor-suppressor function in esophageal epithelium, and its epigenetic silencing may play a pivotal role during esophageal carcinogenesis. Its expression status in biopsies or resected tumors might serve as an index for identifying ESCC patients for whom combined therapeutic modalities would be recommended.
Assuntos
Carcinoma de Células Escamosas/genética , Metilação de DNA , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Receptores do Ácido Retinoico/genética , Azacitidina/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Proliferação de Células , Epigênese Genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Humanos , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Receptores do Ácido Retinoico/metabolismoRESUMO
Anaplastic thyroid cancer (ATC) is one of the most lethal of all human tumors, but cytogenetic information concerning ATC is extremely limited. Using our in-house array-based comparative genomic hybridization and 14 ATC cell lines with further fluorescence in situ hybridization analysis, we demonstrated amplification of the DUSP26 gene, known by another report as MAP kinase phosphatase-8. DUSP26 was overexpressed in ATC cell lines and primary ATC tumor samples. When overexpressed, either exogenously or endogenously, DUSP26 promoted growth of the ATC cells. DUSP26 encodes a protein containing a dual-specificity phosphatase domain that can dephosphorylate itself. DUSP26 effectively dephosphorylates p38 and has a little effect on extracellular signal-regulated kinase in ATC cells. DUSP26 protein formed a physical complex with p38, and promoted survival of ATC cells by inhibiting p38-mediated apoptosis. Our findings suggest that DUSP26 may act as an oncogene in ATC, and might be a useful diagnostic marker and therapeutic target of this disease.
Assuntos
Carcinoma/patologia , Amplificação de Genes , Proteínas Tirosina Fosfatases/genética , Neoplasias da Glândula Tireoide/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Apoptose/genética , Carcinoma/enzimologia , Carcinoma/genética , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células , Clonagem Molecular , Fosfatases de Especificidade Dupla , Humanos , Hibridização in Situ Fluorescente , Fosfatases da Proteína Quinase Ativada por Mitógeno , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , RNA Interferente Pequeno/farmacologia , Neoplasias da Glândula Tireoide/enzimologia , Neoplasias da Glândula Tireoide/genética , Proteínas Quinases p38 Ativadas por Mitógeno/análise , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
To identify target genes for the hemizygous deletions of chromosome 13 that are recurrently observed in malignant gliomas, we performed genome-wide DNA copy-number analysis using array-based comparative genomic hybridization and gene expression analysis using an oligonucleotide-array. The response gene to complement 32 (RGC32) at 13q14.11 was identified as a deletion target, and its expression was frequently silenced in glioma cell lines compared with normal brain. Levels of RGC32 mRNA tended to decrease toward higher grades of primary astrocytomas, especially in tumors with mutations of p53. Expression of RGC32 mRNA was dramatically increased by exogenous p53 in a p53-mutant glioma cell line, and also by endogenous p53 in response to DNA damage in p53+/+ colon-cancer cells, but not in isogenic p53-/- cells. Chromatin immunoprecipitation and reporter assays demonstrated binding of endogenous p53 protein to the promoter region of the RGC32 gene, implying p53-dependent transcriptional activity. Transiently and stably overexpressed RGC32 suppressed the growth of glioma cells, probably owing to induction of G2/M arrest. Immunocytochemical analysis revealed a concentration of RGC32 protein at the centrosome during mitosis. RGC32 formed a protein complex with polo-like kinase 1 and was phosphorylated in vitro. These observations implied a novel mechanism by which p53 might negatively regulate cell-cycle progression by way of this newly identified transcriptional target. Our results provide the first evidence that RGC32 might be a possible tumor-suppressor for glioma, that it is directly induced by p53, and that it mediates the arrest of mitotic progression.
Assuntos
Proteínas de Ciclo Celular/genética , Cromossomos Humanos Par 13/genética , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas de Ciclo Celular/análise , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Imunoprecipitação da Cromatina , Deleção Cromossômica , Dano ao DNA , Fase G2 , Deleção de Genes , Glioma/química , Humanos , Mitose , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Elementos de Resposta , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/metabolismo , Quinase 1 Polo-LikeRESUMO
Homozygous loss in the genomic sequence, a mechanism for inactivating tumor-suppressor genes (TSGs) in cancer, has been used as a tag for the identification of novel TSGs, and array-based comparative genomic hybridization (array-CGH) has a great potential for high-throughput identification of this change. We identified a homozygous loss of the very-low-density lipoprotein receptor (VLDLR) gene (9p24.2) from genome-wide screening for copy-number alterations in 32 gastric cancer (GC) cell lines using array-CGH. Although previous reports demonstrated mRNA or protein expression of VLDLR in various cancers including GC, the association between genomic losses or epigenetic silencing of this gene and carcinogenesis has never been reported before. Homozygous deletion of VLDLR was also seen in primary GCs, albeit infrequently, and about half of GC cell lines showed lost or reduced VLDLR expression. The VLDLR expression was restored in gene-silenced GC cells after treatment with 5-aza 2'-deoxycytidine. According to methylation analyses, hypermethylation of the VLDLR promoter region, which all of GC lines without its expression showed, occurred in some primary GCs. Restoration of VLDLR type I expression in GC cells reduced colony formation. These results suggest that not only the expression of VLDLR but also genetic or epigenetic silencing of this gene may contribute to tumor formation and be involved in gastric carcinogenesis.
Assuntos
Carcinoma/genética , Epigênese Genética , Deleção de Genes , Inativação Gênica , Receptores de LDL/genética , Neoplasias Gástricas/genética , Biópsia , Carcinoma/metabolismo , Carcinoma/cirurgia , Proliferação de Células , Transformação Celular Neoplásica , Cromossomos Humanos Par 9 , Ilhas de CpG , Metilação de DNA , Homozigoto , Humanos , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regiões Promotoras Genéticas , Receptores de LDL/metabolismo , Neoplasias Gástricas/cirurgia , Células Tumorais CultivadasRESUMO
We performed two-color fluorescence in situ hybridization (FISH) on direct touch smears and liquid-based thin-layer (ThinPrep) cytological preparations of endometrial tumors to detect alterations of chromosome 1 and 17 that present with high incidence in endometrial cancers. The DNA probes used for two-color FISH analysis were a combination of the probes designed for 17cen (cCI 17-321) and 17p13.3 (D17S34), and a combination of the probes designed for 1q12 (D1Z1) and 1p36 (cCI1-5335). Numerical or structural alterations of chromosome 1 and/or 17 were detected in 95% (19 of 20 cases) of the direct touch smears obtained from endometrial cancer, while these alterations were also detected in 93% (12 of 13 cases) of samples obtained from grade 1 endometrioid adenocarcinoma cases, including three cases that could not be diagnosed as positive by conventional Papanicolaou cytopathologic staining. Using ThinPrep cytopathologic preparations, numerical or structural abnormalities were found in 26 (90%) and five (100%) cases, respectively, of samples obtained transcervically from 29 endometrial cancer and five atypical endometrial hyperplasia cases. Therefore, two-color FISH may be a useful diagnostic method for endometrial adenocarcinoma and premalignant lesions that demonstrate only slight cellular atypia in conventional cytopathologic preparations.