RESUMO
Purpose Clinically, hypoxia is associated with increased distant metastasis and poor survival in gastric cancer (GC). In this study, we set out from the cellular interaction to further explain the molecular mechanism of invasion in GC cells under hypoxic conditions. Methods Gastric cancer cells were cultured under 1% O2 (hypoxia-cultured gastric cancer cells, HGC) and 20% O2 condition (normoxic-cultured gastric cancer cells, NGC). NGC was co-cultured with HGC-medium. Scrape and Transwell were used to evaluate invasion and migration. Exosomes from GC were extracted by ultracentrifugation. Electron microscopy images, western-blot used to analyze the size distributions and the number of exosomes. Results HGC-medium induced NGC dissociated. Long non-coding RNA (lncRNA) prostate cancer gene expression marker 1 (PCGEM1) was specifically expressed in HGC exosomes. HGC-derived PCGEM1-riched exosomes could promote the invasion and migration of NGC. On the mechanism, PCGEM1 maintained stability and reduced the degradation of SNAI1, which could induce the epithelialmesenchymal transition of GC. Conclusion LncRNA PCGEM1 was overexpressed in GC cells. And part of the PCGEM1 can be encapsulated into exosomes. These exosomes promoted invasion and migration of other GC cells. We considered PCGEM1 might act as a "scaffold" combined with SNAI1 and prompt the invasion and migration of GC (AU)
Assuntos
Humanos , Exossomos/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Hipóxia Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal/fisiologia , Exossomos/ultraestrutura , Invasividade Neoplásica , Metástase Neoplásica , RNA Longo não Codificante , Fatores de Transcrição da Família Snail/genética , Microambiente Tumoral , Regulação para BaixoRESUMO
PURPOSE: Clinically, hypoxia is associated with increased distant metastasis and poor survival in gastric cancer (GC). In this study, we set out from the cellular interaction to further explain the molecular mechanism of invasion in GC cells under hypoxic conditions. METHODS: Gastric cancer cells were cultured under 1% O2 (hypoxia-cultured gastric cancer cells, HGC) and 20% O2 condition (normoxic-cultured gastric cancer cells, NGC). NGC was co-cultured with HGC-medium. Scrape and Transwell were used to evaluate invasion and migration. Exosomes from GC were extracted by ultracentrifugation. Electron microscopy images, western-blot used to analyze the size distributions and the number of exosomes. RESULTS: HGC-medium induced NGC dissociated. Long non-coding RNA (lncRNA) prostate cancer gene expression marker 1 (PCGEM1) was specifically expressed in HGC exosomes. HGC-derived PCGEM1-riched exosomes could promote the invasion and migration of NGC. On the mechanism, PCGEM1 maintained stability and reduced the degradation of SNAI1, which could induce the epithelial-mesenchymal transition of GC. CONCLUSION: LncRNA PCGEM1 was overexpressed in GC cells. And part of the PCGEM1 can be encapsulated into exosomes. These exosomes promoted invasion and migration of other GC cells. We considered PCGEM1 might act as a "scaffold" combined with SNAI1 and prompt the invasion and migration of GC.
Assuntos
Exossomos/metabolismo , RNA Longo não Codificante/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Hipóxia Tumoral , Comunicação Celular , Movimento Celular/genética , Técnicas de Cocultura/métodos , Meios de Cultura/farmacologia , Transição Epitelial-Mesenquimal/fisiologia , Exossomos/ultraestrutura , Humanos , Invasividade Neoplásica , Metástase Neoplásica , RNA Mensageiro/metabolismo , Fatores de Transcrição da Família Snail/genética , Microambiente Tumoral , Regulação para CimaRESUMO
High-grade serous ovarian carcinoma (HGSOC) and basal-like breast cancer (BLBC) share many features including TP53 mutations, genomic instability and poor prognosis. We recently reported that Elafin is overexpressed by HGSOC and is associated with poor overall survival. Here, we confirm that Elafin overexpression is associated with shorter survival in 1000 HGSOC patients. Elafin confers a proliferative advantage to tumor cells through the activation of the MAP kinase pathway. This mitogenic effect can be neutralized by RNA interference, specific antibodies and a MEK inhibitor. Elafin expression in patient-derived samples was also associated with chemoresistance and strongly correlates with bcl-xL expression. We extended these findings into the examination of 1100 primary breast tumors and six breast cancer cell lines. We observed that Elafin is overexpressed and secreted specifically by BLBC tumors and cell lines, leading to a similar mitogenic effect through activation of the MAP kinase pathway. Here too, Elafin overexpression is associated with poor overall survival, suggesting that it may serve as a biomarker and therapeutic target in this setting.
Assuntos
Neoplasias da Mama/genética , Cistadenocarcinoma Seroso/genética , Elafina/genética , Neoplasias Ovarianas/genética , Western Blotting , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células , Cistadenocarcinoma Seroso/metabolismo , Cistadenocarcinoma Seroso/patologia , Elafina/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases/genética , Células MCF-7 , Avaliação de Resultados em Cuidados de Saúde/estatística & dados numéricos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , Modelos de Riscos Proporcionais , Proteômica , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Recent studies suggest that some serous ovarian carcinomas (SOCs) arise from the fallopian tube (FT) epithelium rather than the ovarian surface epithelium. This hypothesis places emphasis on the FT secretory epithelial cell as a cell-of-origin. Herein, we report the development of a novel ex vivo primary human FT epithelium culture system that faithfully recapitulates the in vivo epithelium, as shown by morphological, ultrastructural and immunophenotypic analyses. Mass spectrometry-based proteomics reveal that these cultures secrete proteins previously identified as biomarkers for ovarian cancer. We also use this culture system to study the response of the FT epithelium to genotoxic stress and find that the secretory cells exhibit a distinct response to DNA damage when compared with neighboring ciliated cells. The secretory cells show a limited ability to resolve the damage over time, potentially leaving them more susceptible to accumulation of additional mutagenic injury. This divergent response is confirmed with in situ studies using tissue samples, further supporting the use of this ex vivo culture system to investigate FT epithelial pathobiology. We anticipate that this novel culture system will facilitate the study of SOC pathogenesis, and propose that similar culture systems could be developed for other organ site-specific epithelia.
Assuntos
Biomarcadores Tumorais/análise , Transformação Celular Neoplásica/metabolismo , Cistadenocarcinoma Seroso/patologia , Epitélio/metabolismo , Tubas Uterinas/patologia , Animais , Células Cultivadas , Cistadenocarcinoma Seroso/metabolismo , Dano ao DNA/genética , Dano ao DNA/fisiologia , Feminino , Humanos , Hibridização In Situ , Neoplasias Ovarianas/patologia , OvariectomiaRESUMO
In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a approximately 480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-beta) of the mouseKif1B-beta gene in this region. The KIF1B-beta gene had at least 47 exons, all of which had a classic exon-intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-beta gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-beta in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-beta was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-beta gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-beta gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-beta gene may not be a candidate for tumor suppressor gene of NB.
Assuntos
Cromossomos Humanos Par 1 , Deleção de Genes , Cinesinas/genética , Proteínas do Tecido Nervoso/genética , Neuroblastoma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Neoplasias do Sistema Nervoso Central/genética , Neoplasias do Sistema Nervoso Central/metabolismo , Clonagem Molecular , Análise Mutacional de DNA , DNA de Neoplasias/análise , Homozigoto , Humanos , Cinesinas/biossíntese , Camundongos , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Neuroblastoma/metabolismo , RNA Neoplásico/biossíntese , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Recently, loss of heterozygosity (LOH) studies suggest that more than two tumor suppressor genes lie on the short arm of chromosome 1 (1p) in neuroblastoma (NB). To identify candidate tumor suppressor genes in NB, we searched for homozygous deletions in 20 NB cell lines using a high-density STS map spanning chromosome 1p36, a common LOH region in NB. We found that the 45-kDa subunit of the DNA fragmentation factor (DFF45) gene was homozygously deleted in an NB cell line, NB-1. DFF45 is the chaperon of DFF40, and both molecules are necessary for caspase 3 to induce apoptosis. DFF35, a splicing variant of DFF45, is an inhibitor of DFF40. We examined 20 NB cell lines for expression and mutation of DFF45 gene by reverse transcription (RT)-polymerase chain reaction (PCR) and RT-PCR-single-strand conformation polymorphism. Some novel variant transcripts of the DFF45 gene were found in NB cell lines, but not in normal adrenal gland and peripheral blood. These variants may not serve as chaperons of DFF40, but as inhibitors like DFF35, thus disrupting the balance between DFF45 and DFF40. No mutations of the DFF45 gene were found in any NB cell line, suggesting that the DFF45 is not a tumor suppressor gene for NB. However, homozygous deletion of the DFF45 gene in the NB-1 cell line may imply the presence of unknown tumor suppressor genes in this region.
Assuntos
Cromossomos Humanos Par 1 , Deleção de Genes , Neuroblastoma/genética , Proteínas/genética , Processamento Alternativo , Proteínas Reguladoras de Apoptose , Caspase 3 , Caspases/metabolismo , Fragmentação do DNA , Homozigoto , Humanos , Perda de Heterozigosidade , Mutação , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sitios de Sequências Rotuladas , Células Tumorais CultivadasRESUMO
We performed expression, mutation, loss of heterozygosity (LOH) and fluorescence in situ hybridization (FISH) analyses of the p73 gene in neuroblastomas (NBs). Reverse transcription-polymerase chain reaction (RT-PCR) using primers which can detect both the p73alpha and p73beta transcripts was performed on 30 fresh NBs and 22 NB cell lines. Aberrant expression of the p73 gene was found in 4 (25%) of 16 primary tumors found by mass screening and in 10 (71.4%) of 14 primary tumors found clinically. The rates of expression in these two types of tumors were significantly different (p=0.026, Fisher's exact test). The incidence of aberrant expression of the p73 gene was significantly higher in stage IV patients than in stages I, II, III plus IVS patients (p=0.0236, Fisher's exact test). No homozygous deletions or rearrangements of the p73 gene were found in any samples examined. In addition to the polymorphism in exon 2, a silent mutation (codon 336 GCC/GCT) was found in one primary tumor. LOH of the p73 gene was detected in 5 (15%) of 33 primary NBs using PCR-LOH analysis. FISH analysis showed that all 17 NB cell lines used in this study revealed allelic loss of the p73 gene, while most of them expressed the p73 gene. These results suggest that the p73 gene is not monoallelically expressed in NB. We conclude that the p73 gene is less involved in the development but involved in the progression of neuroblastoma.
Assuntos
Proteínas de Ligação a DNA/genética , Genes Supressores de Tumor/genética , Neuroblastoma/genética , Proteínas Nucleares/genética , Primers do DNA/química , DNA de Neoplasias/análise , Proteínas de Ligação a DNA/metabolismo , Progressão da Doença , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Perda de Heterozigosidade , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Proteínas Nucleares/metabolismo , Mutação Puntual , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteína Tumoral p73 , Proteínas Supressoras de TumorRESUMO
Both p16 and p15, encoded by genes located on chromosome 9p21, are inhibitors of cyclin-dependent kinases 4/6 (CDK4/6) and upstream regulators of RB function, and set up the RB/p16 tumor suppressive pathway, which is abrogated frequently in human neoplasms, either through inactivation of the RB or p16 tumor-suppressor protein, or alteration of the cyclin D1 or CDK4 oncoproteins. In hematological malignancies, deletion of p16/p15 locus has been shown to be highly specific to lymphoid malignancies, and more particularly to T-cell acute lymphoblastic leukemia (T-ALL). However, in the other subsets of ALL, deletions of p16 and p15 are relatively rare events. To investigate whether these genes are inactivated by methylation of the 5' CpG islands, we examined 35 leukemia cell lines and 29 childhood acute myeloid leukemia (AML) patients by Southern blot, polymerase chain reaction (PCR) and Western blot analyses. We found methylation of p16 in 12 (50%) of 24 ALL cell lines, 5 (50%) of 10 AML cell lines without homozygous deletion of p16, and 11 (38%) of 29 AML patients. Those leukemia cell lines subjected to p16 methylation were found to have lost p16 protein expression. The p15 gene was methylated in 10 (34%) of 29 ALL cell lines, 6 (60%) of 10 AML cell lines without homozygous deletion of p15, and 15 (52%) of 29 AML patients. These results revealed the frequent methylation of p16 and p15 genes in B-ALL and AML despite a low frequency of p16 and p15 deletions and mutations in these leukemias. In the study for expression of RB protein, we found no expression of RB in 4 of 16 leukemia cell lines. Inactivation of the p16 gene was found in all the cell lines with expression of RB. Neither amplification nor rearrangement of cyclin D1 gene was found in any cell lines. These results suggest that inactivation of p16 and p15 genes is one of the most common genetic events in acute leukemia, and plays an important role for the RB/p16 pathway in the pathogenesis of acute leukemia.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina/biossíntese , Metilação de DNA , DNA de Neoplasias/genética , Regulação Leucêmica da Expressão Gênica , Genes do Retinoblastoma , Genes p16 , Leucemia Mieloide/genética , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteína do Retinoblastoma/biossíntese , Proteínas Supressoras de Tumor , Doença Aguda , Linfoma de Burkitt/genética , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Proteínas de Transporte/biossíntese , Ilhas de CpG , Ciclina D1/biossíntese , Ciclina D1/genética , Inibidor de Quinase Dependente de Ciclina p15 , DNA de Neoplasias/química , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Perda de Heterozigosidade , Técnicas de Sonda Molecular , Proteínas de Neoplasias/biossíntese , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Deleção de Sequência , Células Tumorais CultivadasRESUMO
Chromosomal aberrations and inactivation of tumour suppressor genes are frequent in acute leukaemia. To determine whether the FHIT gene is involved in the development of leukaemia, we examined the FHIT transcript in 65 leukaemia cell lines, 5 fresh acute leukaemia patients at diagnosis and in complete remission, normal peripheral blood lymphocytes obtained from 14 healthy volunteers and Epstein-Barr (EB) virus transformed 5 B-cell lines (EB-lines), using nested reverse transcription-polymerase chain reaction and direct sequencing. The transcripts were classified into 4 patterns: pattern I revealed the normal transcripts only, pattern II the altered transcripts in addition to the normal transcripts, pattern III the altered transcripts without the normal transcripts and pattern IV an absence of normal and altered FHIT transcripts. Nineteen cell lines were classified as pattern I, 32 as pattern II, 2 as pattern III and 12 as pattern IV. The frequency of loss of FHIT expression (pattern III or IV) varied in each type of leukaemia cell line; the order ranked from the highest incidence was acute myeloid leukaemia (AML), T-cell acute lymphoblastic leukaemia (T-ALL), B-precursor ALL, B-ALL, and chronic myeloid leukaemia (CML). No genomic rearrangement was found in any samples examined. All of 5 patients showed same pattern II FHIT transcripts at 2 different stages of the disease. All normal peripheral blood lymphocytes and EB-lines were classified as pattern I or II. Our results suggested that patterns III and IV of FHIT transcripts might be associated with the development of a subset of leukaemia, while pattern II which has so far been reported as an aberrant transcript in varieties of malignant tumours might not be associated with leukaemogenesis.