RESUMO
5-Fluorouracil (5-FU) is one of the most commonly used chemotherapeutic agents in colon cancer treatment, but has a narrow therapeutic index limited by its toxicity. Melatonin exerts antitumor activity in various cancers, but it has never been combined with 5-FU as an anticolon cancer treatment to improve the chemotherapeutic effect of 5-FU. In this study, we assessed such combinational use in colon cancer and investigated whether melatonin could synergize the antitumor effect of 5-FU. We found that melatonin significantly enhanced the 5-FU-mediated inhibition of cell proliferation, colony formation, cell migration and invasion in colon cancer cells. We also found that melatonin synergized with 5-FU to promote the activation of the caspase/PARP-dependent apoptosis pathway and induce cell cycle arrest. Further mechanism study demonstrated that melatonin synergized the antitumor effect of 5-FU by targeting the PI3K/AKT and NF-κB/inducible nitric oxide synthase (iNOS) signaling. Melatonin in combination with 5-FU markedly suppressed the phosphorylation of PI3K, AKT, IKKα, IκBα, and p65 proteins, promoted the translocation of NF-κB p50/p65 from the nuclei to cytoplasm, abrogated their binding to the iNOS promoter, and thereby enhanced the inhibition of iNOS signaling. In addition, pretreatment with a PI3K- or iNOS-specific inhibitor synergized the antitumor effects of 5-FU and melatonin. Finally, we verified in a xenograft mouse model that melatonin and 5-FU exerted synergistic antitumor effect by inhibiting the AKT and iNOS signaling pathways. Collectively, our study demonstrated that melatonin synergized the chemotherapeutic effect of 5-FU in colon cancer through simultaneous suppression of multiple signaling pathways.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias do Colo/patologia , Fluoruracila/farmacologia , Melatonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Sinergismo Farmacológico , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Nus , Microscopia Confocal , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) has a significant contributory role in the development of three major human neoplastic or lymphoproliferative diseases: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD). These diseases are associated with chromosomal instability, a hallmark of human cancer. The latency-associated nuclear antigen (LANA) encoded by KSHV plays a key role in regulating a number of cellular pathways critical for oncogenesis. KSHV LANA alone can induce the development of B-cell hyperplasia and lymphoma in mice expressing LANA. LANA also induces chromosomal instability, thus promoting oncogenesis. However, the precise mechanism underlying LANA-mediated chromosomal instability remains uncharted. Here we report that LANA promoted the induction of chromosomal instability and the formation of micronuclei and multinucleation through its interaction with one of the critical spindle checkpoint proteins, Bub1, and the resulting degradation of Bub1. This interaction occurs through the Knl and kinase domains of Bub1, identified as important for stability and degradation. These results suggest that LANA can dysregulate Bub1 activity, which leads to aberrant chromosome replication and aneuploidy, thus contributing to KSHV-mediated oncogenesis. IMPORTANCE: This work represents the first set of results identifying a novel mechanism by which LANA, a latency-associated antigen encoded by KSHV, can induce the degradation of Bub1, a spindle checkpoint protein that is important for spindle checkpoint signaling and chromosome segregation. The downregulation of Bub1 mediated by LANA resulted in chromosomal instability, a hallmark of cancer. We further investigated the specific domains of Bub1 that are required for the interaction between LANA and Bub1. The results demonstrated that the Knl and kinase domains of Bub1 are required for the interaction between LANA and Bub1. In addition, we also investigated the mechanism by which LANA promoted Bub1 degradation. Our results showed that LANA interacted physically with the anaphase-promoting complex (APC/C), thus promoting the degradation of Bub1 in a ubiquitin-dependent process.
Assuntos
Antígenos Virais/metabolismo , Instabilidade Cromossômica , Herpesvirus Humano 8/fisiologia , Linfoma de Efusão Primária/virologia , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Sarcoma de Kaposi/virologia , Animais , Western Blotting , Humanos , Imunoprecipitação , Linfoma de Efusão Primária/genética , Linfoma de Efusão Primária/metabolismo , Camundongos , Micronúcleos com Defeito Cromossômico , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Células Tumorais Cultivadas , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
Aberrant expression of Aurora A kinase has been frequently implicated in many cancers and contributes to chromosome instability and phosphorylation-mediated ubiquitylation and degradation of p53 for tumorigenesis. Previous studies showed that p53 is degraded by Kaposi's sarcoma herpesvirus (KSHV) encoded latency-associated nuclear antigen (LANA) through its SOCS-box (suppressor of cytokine signaling, LANA(SOCS)) motif-mediated recruitment of the EC(5)S ubiquitin complex. Here we demonstrate that Aurora A transcriptional expression is upregulated by LANA and markedly elevated in both Kaposi's sarcoma tissue and human primary cells infected with KSHV. Moreover, reintroduction of Aurora A dramatically enhances the binding affinity of p53 with LANA and LANA(SOCS)-mediated ubiquitylation of p53 which requires phosphorylation on Ser215 and Ser315. Small hairpin RNA or a dominant negative mutant of Aurora A kinase efficiently disrupts LANA-induced p53 ubiquitylation and degradation, and leads to induction of p53 transcriptional and apoptotic activities. These studies provide new insights into the mechanisms by which LANA can upregulate expression of a cellular oncogene and simultaneously destabilize the activities of the p53 tumor suppressor in KSHV-associated human cancers.
Assuntos
Herpesvirus Humano 8/genética , Proteínas Serina-Treonina Quinases/genética , Proteína Supressora de Tumor p53/genética , Antígenos Virais/genética , Antígenos Virais/metabolismo , Aurora Quinases , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica , Herpesvirus Humano 8/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Leucócitos Mononucleares/virologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Transcrição Gênica , Transfecção , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação , Regulação para CimaRESUMO
The Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential latent antigens for Epstein-Barr virus (EBV)-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103) is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs). Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities.
Assuntos
Antígenos Virais/metabolismo , Apoptose/fisiologia , Proteína DEAD-box 20/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Antígenos Virais/genética , Linfócitos B/metabolismo , Linfócitos B/virologia , Western Blotting , Linhagem Celular Tumoral , Proteína DEAD-box 20/genética , Infecções por Vírus Epstein-Barr/genética , Antígenos Nucleares do Vírus Epstein-Barr , Imunofluorescência , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Herpesvirus Humano 4/genética , Humanos , Imunoprecipitação , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Proteína Supressora de Tumor p53/genéticaRESUMO
The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) is critical for segregation of viral episomes to progeny nuclei and allows for maintenance of the viral genome in newly divided daughter cells. LANA binds to KSHV terminal repeat (TR) DNA and simultaneously associates with chromatin-bound cellular proteins. This process tethers the viral episomes to host chromosomes. However, the mechanism of tethering is complex and involves multiple protein-protein interactions. Our previous proteomics studies which showed the association of LANA with centromeric protein F (CENP-F) prompted us to further study whether LANA targets centromeric proteins for persistence of KSHV episomes during cell division. Here we show that LANA colocalized with CENP-F as speckles, some of which are paired at centromeric regions of a subset of chromosomes in KSHV-infected JSC-1 cells. We also confirm that both the amino and carboxy termini of LANA can bind to CENP-F. Moreover, LANA associated with another kinetochore protein, Bub1 (budding uninhibited by benzimidazole 1), which is known to form a complex with CENP-F. Importantly, we demonstrated the dynamic association of LANA and Bub1/CENP-F and the colocalization between Bub1, LANA, and the KSHV episome tethered to the host chromosome using fluorescence in situ hybridization (FISH). Knockdown of Bub1 expression by lentivirus-delivered short hairpin RNA (shRNA) dramatically reduced the number of KSHV genome copies, whereas no dramatic effect was seen with CENP-F knockdown. Therefore, the interaction between LANA and the kinetochore proteins CENP-F and Bub1 is important for KSHV genome tethering and its segregation to new daughter cells, with Bub1 potentially playing a more critical role in the long-term persistence of the viral genome in the infected cell.
Assuntos
Antígenos Virais/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/fisiologia , Cinetocoros/virologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Antígenos Virais/química , Antígenos Virais/genética , Sequência de Bases , Linhagem Celular , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/genética , Técnicas de Silenciamento de Genes , Genoma Viral , Herpesvirus Humano 8/patogenicidade , Interações Hospedeiro-Patógeno , Humanos , Técnicas In Vitro , Cinetocoros/metabolismo , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/genética , Mitose , Modelos Biológicos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Plasmídeos/genética , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/genéticaRESUMO
Survivin is a master regulator of cell proliferation and cell viability and is highly expressed in most human tumors. The molecular network linked to survivin expression in tumors has not been completely elucidated. In this study, we show that latency-associated nuclear antigen (LANA), a multifunctional protein of Kaposi's sarcoma-associated herpesvirus (KSHV) that is found in Kaposi's sarcoma tumors, upregulates survivin expression and increases the proliferation of KSHV-infected B cells. Analysis of pathway-specific gene arrays showed that survivin expression was highly upregulated in BJAB cells expressing LANA. The mRNA levels of survivin were also upregulated in HEK 293 and BJAB cells expressing LANA. Similarly, protein levels of survivin were significantly higher in LANA-expressing, as well as KSHV-infected, cells. Survivin promoter activity assays identified GC/Sp1 and p53 cis-acting elements within the core promoter region as being important for LANA activity. Gel mobility shift assays revealed that LANA forms a complex with Sp1 or Sp1-like proteins bound to the GC/Sp1 box of the survivin promoter. In addition, a LANA/p53 complex bound to the p53 cis-acting element within the survivin promoter, indicating that upregulation of survivin expression can also occur through suppression of p53 function. Furthermore, immunohistochemistry analyses revealed that survivin expression was upregulated in KSHV-associated Kaposi's sarcoma tissue, suggesting that LANA plays an important role in the upregulation of survivin expression in KSHV-infected endothelial cells. Knockdown of survivin expression by lentivirus-delivered small hairpin RNA resulted in loss of cell proliferation in KSHV-infected cells. Therefore, upregulation of survivin expression in KSHV-associated human cells contributes to their proliferation.
Assuntos
Antígenos Virais/metabolismo , Proliferação de Células , Herpesvirus Humano 8/metabolismo , Linfoma de Células B/fisiopatologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/metabolismo , Sarcoma de Kaposi/fisiopatologia , Regulação para Cima , Antígenos Virais/genética , Linhagem Celular , Regulação da Expressão Gênica , Herpesvirus Humano 8/genética , Humanos , Proteínas Inibidoras de Apoptose , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/virologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Sarcoma de Kaposi/virologia , Fator de Transcrição Sp1/genética , Fator de Transcrição Sp1/metabolismo , Survivina , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Studies have revealed that Epstein-Barr virus (EBV) infection, genetic aberration, and environmental factors are of importance in the development of nasopharyngeal carcinoma (NPC), although the definite mechanism remains to be fully elucidated. The aim of our study is to investigate using tissue microarray analysis whether differential expression of EBV-encoded small RNA-1 (EBER-1) and several tumor-related genes were associated with NPC carcinogenesis. Immunohistochemistry and in situ hybridization were performed on tissue microarrays containing 148 NPCs and 164 noncancerous nasopharyngeal epithelia (NPE) with different morphologic features. We found that overexpressions of EBER-1 hybridization signals, p53, p21ras, and bcl-2 proteins and loss expressions of p16 and p27 proteins were significantly increased in NPC tissues compared with normal NPE and hyperplastic NPE (P = .001). The overexpressions of EBER-1 and p53 (P < .001) and the loss expressions of P16 (P < .001) and P27 (P = .005) were also significantly higher and more frequently observed in NPC than in dysplastic NPE. The positive expression of EBER-1 hybridization signals in NPC had significant associations with overexpressions of p53 (P < .001), p21ras (P = .041), and bcl-2 proteins (P < .001) and loss expression of p16 protein (P = .001). Further analysis confirmed that the abnormal expression of p53, p16, and p27 proteins occurred in the earliest stage of nasopharyngeal epithelial carcinogenesis. In the final logistic regression analysis model, the positive hybridization signals of EBER-1 and the abnormal expression of p53, p16, and p27 proteins were independent contributions for nasopharyngeal carcinogenesis, and EBER-1 was the most significant, independent predictor of nasopharyngeal carcinogenesis (hazard ratio = 13.412, 95% confidence interval 6.179-29.111, P < .001). In conclusion, EBV infection, together with overexpressions of p53, and loss expressions of p16 and p27 proteins are involved in the multistep process of human nasopharyngeal epithelial carcinogenesis.
Assuntos
Adenocarcinoma/virologia , Neoplasias Nasofaríngeas/virologia , Proteínas de Neoplasias/metabolismo , RNA Viral/análise , Análise Serial de Tecidos/métodos , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Humanos , Hiperplasia , Imuno-Histoquímica , Hibridização In Situ , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Proteínas de Neoplasias/genéticaRESUMO
OBJECTIVE: To study the suppressive effect of LRRC4 gene on human glioma U251 cells and further investigate its biological functions. METHODS: H&E, DNA and AgNORs stainings were performed on LRRC4-transfected U251 cells, mock-transfected U251 cells and non-transfected U251 cells, respectively. Quantitative analysis including cell morphometry, DNA content, DNA ploidy, silver stained argyrophilic nucleolar organizer regions (AgNORs) were investigated by image analysis. Flow cytometry was employed to determine the difference of cell cycle distribution and MTT staining was used to elucidate the activity of the LRRC4-transfected U251 cells. RESULTS: The morphological cell parameters such as area, perimeter and diameter, DNA content, chromosomal aneupoloidy, mean area of AgNORs particles and mean nucleus area of the LRRC4-transfected U251 cells were remarkably decreased compared to those of the mock-transfected and non-transfected U251 cells (P < 0.05, P < 0.01). Meanwhile, significant accumulation of cells in G(0)/G(1) phase but decrease of cells in S and G(2)/M phase, was observed in transfected U251 cells compared to those of the mock-transfected and non-transfected U251 cells (P < 0.05, P < 0.01). MTT staining showed that proliferation activity of both the mock- and non-trasfected U251 cells was significantly higher than that of the U251 cells transfected with LRRC4 gene (P < 0.01). CONCLUSION: LRRC4 gene might be involved in tumor suppression by restraining DNA synthesis and the nucleoli organizer regions-associated proteins, keeping the cell cycles in phase G(0)/G(1) and reducing proliferation activity of the glioma cells. Morphometry combined with other techniques such as flow cytometry and MTT staining can well elucidate the biological function of novel genes.
Assuntos
Neoplasias Encefálicas/genética , Genes Supressores de Tumor/fisiologia , Glioblastoma/genética , Proteínas do Tecido Nervoso/genética , Neoplasias Encefálicas/patologia , Cromossomos Humanos Par 7 , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Humanos , Proteínas do Tecido Nervoso/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
PURPOSE: To investigate cloning, expression, and mutation analysis of the putative candidate tumor suppressor gene related with nasopharyngeal carcinoma (NPC). METHODS: We studied the expression profiles in the NPC cell line HNE(1) with the normal nasopharyngeal epithelial cell as control by using cDNA array representing 11,000 cDNA clusters. EST W95442 was found down-regulated in HNE(1). Subsequently, the corresponding gene sequence including this EST was established by cDNA cloning and the RACE (rapid amplification of cDNA end) procedure. The expression pattern of this gene was examined by using Northern blot analysis in various human tissues. Furthermore, we screened the mutations of the coding sequence of the gene using reverse transcription-polymerase chain reaction and single-strand conformation polymorphisms (RT-PCR-SSCP) as well as direct sequencing analysis. RESULTS: A novel gene (GenBank accession No. AF462348) was cloned and named NOR(1) standing for oxidored-nitro domain-containing protein 1 (Human Gene Nomenclature Committee-approved symbol). Northern blot analysis revealed that the NOR(1) gene had two transcripts (1.2 kb, 1.6 kb), and expressed ubiquitously in human tissues. Moreover, a Glu58Gly mutation in the exon 1 of NOR(1) was detected in two of 25 NPC biopsies. CONCLUSIONS: We cloned a novel gene NOR(1), and the Glu58Gly polymorphism of NOR(1) may be involved in the development and/or progression of NPC suggesting that NOR(1) could be a candidate tumor repressor gene related with NPC.
Assuntos
Neoplasias Nasofaríngeas/genética , Proteínas/genética , Sequência de Aminoácidos , Sequência de Bases , Cromossomos Humanos Par 9 , Clonagem Molecular , Análise Mutacional de DNA , Regulação para Baixo , Expressão Gênica , Humanos , Proteínas de Membrana Transportadoras , Mutação , Células Tumorais CultivadasRESUMO
To get genotype and allele frequency distributions of seven short tandem repeat (STR) loci of chromosome 9p,D9S288,D9S157,D9S1748,D9S171,D9S161,D9S1817 and D9S1805 in Chinese Han population in Hunan area,blood samples were collected from the random Han individual in Hunan and the whole genomic DNA was extracted.STR loci were amplified by multiplex-PCR technique and genotyped by ABI 377 sequencer.Seventy-five alleles were detected,with frequencies ranging from 0.002 to 0.800,and constituted 243 genotypes. All the seven loci met Hardy-Weinberg equilibrium. The statistical analysis of seven STR loci showed H(heterozygosity) ranging from 0.347 to 0.844,DP(discrimination power) ranging from 0.346 to 0.841,PPE(probabilities of paternity exclusion) ranging from 0.308 to 0.738 and PIC(polymorphic information content) ranging from 0.328 to 0.822. The result indicated that there was a significant difference between Han ethnic group and the white and the black.
RESUMO
Recently, the UBAPl gene, a putative nasopharyngeal carcinoma (NPC) related gene, which is located at human chromosome 9p21-22 where loss of heterozygosity frequently occurs in NPC, was cloned. The present study aimed to explore the purification approach to UBAP1 protein and its expression pattern in NPC with tissue microarray. The full length coding sequence of UBAP1 was subcloned into a prokaryotic expression vector, pGEX-4T-2, and expressed in Escherichia coli as a GST-fusion protein. With modification of the purification method, GST-UBAP1 fusion protein achieved a high level of purity. The New Zealand rabbit was immunized with the purified fusion protein to prepare polyclonal antiserum following standard protocols. With this antiserum, high-throughput analysis of UBAP1 protein expression using immunohistochemistry was performed on self-made tissue microarrays consisting of 316 nasopharyngeal specimens from 148 NPC and 168 non-cancerous nasopharyngeal epithelia with different morphological features. Consequently, we found that there was a significant decrease in the percentage of positive expression in all NPC on protein levels for UBAP1 (23%), compared to that of the non-NPC (89%) (P<0.01). The result suggests that UBAP1 might be a potential effective diagnosis candidate for NPC and decreased expression of UBAP1 protein is a possible point of dysfunction along the pathogenesis pathway for NPC that may contribute to malignant transformation.
Assuntos
Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/isolamento & purificação , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/metabolismo , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Transformação Celular Neoplásica/genética , Regulação para Baixo/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neoplasias Nasofaríngeas/etiologia , Análise Serial de Proteínas , CoelhosRESUMO
BACKGROUND & OBJECTIVE: NOR(1)is a good candidate of tumor suppressor/susceptibility gene associated with nasopharyngeal carcinoma. This study was designed to construct the prokaryotic expression vector and to investigate the expression of nitroreductase gene NOR(1)in Escherichia coli and to purify expressed product. METHODS: Total RNA was subtracted from normal nasopharyngeal carcinoma tissue. The full length of NOR(1)gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHIand XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI,then the NOR(1)gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR(1)was identified by sequencing and digested with restriction enzymes. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express GST fusion protein. The result was confirmed by Western blot analysis and the purified targeted protein was obtained by affinity chromatography. RESULTS: The 1.25kb NOR(1)gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). It existed not only in supernatant but also in precipitation of broken bacteria. The result was confirmed by Western blot analysis,and the purified targeted protein was obtained by affinity chromatography. CONCLUSION: The successes in construction of expression vector of NOR(1), expression and purification of GST/NOR(1)fusion protein make it possible to prepare for the polyantibodies for NOR(1).
Assuntos
Nitrorredutases/biossíntese , Clonagem Molecular , Escherichia coli/genética , Humanos , Nitrorredutases/genética , Nitrorredutases/isolamento & purificação , Proteínas Recombinantes/biossínteseRESUMO
BACKGROUND & OBJECTIVE: NASG gene, a tissue-specific gene of human nasopharyngeal epithelium was isolated by suppression subtractive hybridization. This study was designed to analyze splicing variants in NASG 3'untranslated region (UTR) and its expression profiling in multiple cancer tissues. METHODS: The PCR primers were designed in NASG 3'UTR around the splicing variants and reverse transcription-polymerase chain reaction (RT-PCR) was performed. The PCR products were separated and sequenced. The expression patterns of NASG were detected by RT-PCR among nasopharyngeal carcinoma (NPC) cell line HNE1, primary human embryo nasopharyngeal epithelial cells, NPC biopsies, and normal adult nasopharyngeal epithelial tissues. Its expression profiling in multiple cancer tissues were tested by cancer profiling array hybridization. RESULTS: There were three splicing variants in NASG 3'UTR. NASG was identified to be down-regulated in NPC cell line HNE1 and 71% of the NPC biopsies, but up-regulated in 25% lung of the cancer biopsies, and not express in other cancer tissues and normal tissues. CONCLUSION: There were three splicing variants in NASG 3'UTR. Its abnormal expression may be an important molecular event in NPC and lung cancer.
Assuntos
Regiões 3' não Traduzidas/genética , Processamento Alternativo , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/genética , Neoplasias Nasofaríngeas/metabolismo , Proteínas de Neoplasias/genética , Fosfoproteínas/genética , Adulto , Sequência de Bases , DNA de Neoplasias/análise , Regulação para Baixo , Glicoproteínas/metabolismo , Humanos , Dados de Sequência Molecular , Neoplasias Nasofaríngeas/genética , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Análise de Sequência de DNARESUMO
Suppression subtractive hybridization (SSH) was performed for isolation of tissue-specific genes in nasopharyngeal epithelial tissue, by use of cDNAs from human adult nasopharyngeal epithelial tissue as tester and mixed cDNAs from esophagus, lung, liver, heart, stomach, spleen, skeletal muscle, kidney, and skin as drivers. Fourteen differentially expressed genes in nasopharyngeal epithelial tissue were obtained. Among these genes, LPLUNC1 and SPLUNC1 were confirmed to be specifically expressed in nasopharyngeal epithelial tissue and the trachea. A novel transcript of SPLUNC1, which we designate NASG, was found. We also combined SSH and cDNA microarray hybridization to identify genes whose expressions were altered in nasopharyngeal carcinoma (NPC). We used NPC cell line HNE1 and primary human embryo nasopharyngeal epithelial cells in one SSH experiment, and NPC biopsies and normal adult nasopharyngeal epithelial tissue in another. Some 1,200 SSH inserts from four subtractive cDNA libraries were arrayed onto nylon membranes by use of robotic printing. Differential gene expression was verified by hybridizing of the membranes with radioactively labeled first-strand cDNA from NPC cell line HNE1, primary human embryo nasopharyngeal epithelial cells, NPC biopsies, and normal adult nasopharyngeal epithelial tissue. Seventeen differentially expressed genes in NPC were obtained. Among these genes, we identified SPLUNC1 and LPLUNC1 to be down-expressed in NPC biopsies (34/48, 33/48).