Biomedical subjects

Yohko Nakamura

Publications and source records attributed to Yohko Nakamura.

11 recordsLinked to original sources

PubMed2006

Relationship of DDX1 and NAG gene amplification/overexpression to the prognosis of patients with MYCN-amplified neuroblastoma.

PURPOSE: Amplification of the MYCN gene strongly correlates with advanced stage, rapid tumor progression and poor prognosis in neuroblastoma (NB). Several genes in the MYCN amplicon, including the DEAD box polypeptide 1 (DDX1) gene, and neuroblastoma-amplified gene (NAG gene), have been found to be frequently co-amplified with MYCN in NB. The aim of this study was to clarify the prognostic significance of the co-amplification or overexpression of DDX1 and NAG with MYCN. PROCEDURE: The gene copy numbers and mRNA expression levels of MYCN, DDX1, and NAG in 113 primary NBs were determined by the real-time quantitative polymerase chain reaction or quantitative reverse transcriptase/polymerase chain reaction assay. The relationships between gene co-amplification/overexpression status and stage, age at diagnosis, and overall survival were analyzed. RESULTS: For evaluating the frequency of DDX1 and NAG co-amplification, it proved appropriate to discriminate NBs with <40 copies of MYCN amplification from those with > or =40 copies of MYCN (DDX1, p = 0.00058; NAG, p = 0.0242, chi(2) for independence test). In patients with MYCN-amplified NB aged > or =18 months, those with tumor with enhanced DDX1 expression and low-NAG expression showed a significantly better outcome than those with low-DDX1 expression or enhanced NAG expression (p = 0.0245, log-rank test). None of the gene expression statuses had a significant relation to disease stage or survival for patients <18 months old. No relationship between any gene co-amplification status and disease stage, age at diagnosis, or overall survival was found. CONCLUSIONS: Our findings suggest that there may be a subset of NB in which enhanced DDX1 and low-NAG expression consequent to DDX1 co-amplification without NAG amplification contributes to susceptibility to intensive therapy. A larger study using an age cut-off of 18 months will be required.

Adolescent↗

PubMed2006

High expression of N-acetylglucosaminyltransferase V in favorable neuroblastomas: Involvement of its effect on apoptosis.

Neuroblastoma (NBL), derived from the sympathetic precursor cells, is one of the most common pediatric solid tumors. The expression of N-acetylglucosaminyltransferase V and IX (GnT-V and GnT-IX) mRNA in 126 primary NBLs were quantitatively analyzed and higher expression levels of GnT-V were found to be associated with favorable stages (1, 2 and 4s). Conversely, the downregulation of GnT-V expression by small interfering RNA resulted in a decrease in the susceptibility to cell apoptosis induced by retinoic acid in NBL cells accompanied by morphological change. These results suggest that GnT-V is associated with prognosis by modulating the sensitivity of NBLs to apoptosis.

Animals↗

PubMed2006

Aberrant methylation of RASGRF2 and RASSF1A in human non-small cell lung cancer.

Aberrant methylation of promoter CpG that causes silencing of tumor suppressor genes (TSGs) may play a key role in the carcinogenesis of many cancer types. RASSF1A, regarded as a TSG, has been extensively studied in lung cancer and other malignant tumors, whereas RASGRF2 has only been reported to possibly play a role in the pathogenesis of pancreatic cancer cell lines. The aims of our study were to i) determine the methylation profile of RASGRF2 and ii) compare the methylation profiles of RASGRF2 with RASSF1A in lung cancer. We examined RASGRF2 expression by reverse transcription PCR and aberrant methylation of RASGRF2 by methylation-specific PCR in lung cancer cell lines. Loss of RASGRF2 expression was presented in 36% lung cancer cell lines while aberrant methylation of RASGRF2 was present in 30% (3/10) non-small cell lung cancer (NSCLC) cell lines and in 25% (1/4) small cell lung cancer (SCLC) cell lines. The concordance between loss of expression and aberrant methylation of RASGRF2 was 86% (12/14). RASGRF2 expression was restored after treatment with the demethylating agent, 5-aza-2'-deoxycytidine in all four cell lines tested that downregulated RASGRF2 expression. Among primary NSCLC, RASGRF2 and RASSF1A methylation was observed in 34% (39/114) and 39% (44/114) of cases respectively, while it was observed in only 7% (4/57) and none of the corresponding non-malignant lung tissue. There is no correlation between RASGRF2 and RASSF1A methylation status. Both RASGRF2 and RASSF1A methylation did not associate with clinical characteristics. Frequent methylation and silencing of RASGRF2 in tumor cells may play an important role, different from that of RASSF1A, in the carcinogenesis of NSCLC.

Aged↗

PubMed2005

Expression profiling using a tumor-specific cDNA microarray predicts the prognosis of intermediate risk neuroblastomas.

To predict the prognosis of neuroblastoma patients and choose a better therapeutic protocol, we developed a cDNA microarray carrying 5340 genes obtained from primary neuroblastomas and examined 136 tumor samples. We made a probabilistic output statistical classifier that provided a high accuracy in prognosis prediction (89% at 5 years) and a highly reliable method to validate it. Kaplan-Meier analysis indicated that the patients in an intermediate group defined by existing markers are divided by microarray into two further groups with 5 year survivals for 36% and 89% of patients (p < 10(-4)), i.e., with unfavorably and favorably predicted neuroblastomas, respectively. According to these results, we developed a gene subset chip for a clinical tool, for which our classifier exhibited 88% prediction accuracy.

Algorithms↗

PubMed2005

Aberrant methylation of FBN2 in human non-small cell lung cancer.

FBN2, a large modular extracellular matrix glycoprotein, is known to be a key component of human elastic fiber. A loss of FBN2 expression due to promoter methylation was recently identified in pancreatic cancer. We examined FBN2 expression by reverse transcription PCR and aberrant methylation of FBN2 by methylation specific PCR in lung cancer cell lines. Aberrant methylation of FBN2 was present in 55% (6 of 11) of non-small cell lung cancer (NSCLC) cell lines, but it absent in small cell lung cancer cell lines. The concordance between loss of expression and aberrant methylation of FBN2 was 88% (14 of 16) in the cell lines. FBN2 expression was restored after treatment with the demethylating agent, 5-aza-2'-deoxycytidine in all six cell lines tested that lacked FBN2 expression. Among primary NSCLC, 49% (62/126) of cases had FBN2 methylation, but only 7% (5/69) of the corresponding nonmalignant lung tissues had it. Although FBN2 methylation was detected even in patients with early stage disease, it occurred frequently in large tumors (p=0.022), with nodal metastasis (p=0.037), or with advanced stages of NSCLC (p=0.014). Methylation and silencing of FBN2 in tumor cells may play an important role in carcinogenesis, invasion, and metastasis of NSCLC.

Aged↗

PubMed2004

Low expression of human tubulin tyrosine ligase and suppressed tubulin tyrosination/detyrosination cycle are associated with impaired neuronal differentiation in neuroblastomas with poor prognosis.

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups. However, the precise mechanism underlying this phenomenon remains largely unknown. To understand the molecular and genetic bases of neuroblastoma, we have generated its cDNA libraries and identified a human ortholog of tubulin tyrosine ligase gene (hTTL/Nbla0660) as a differentially expressed gene at high levels in a favorable subset of the tumor. Tubulin is subjected to several types of evolutionarily conserved posttranslational modification, including tyrosination and detyrosination. Tubulin tyrosine ligase catalyzes ligation of the tyrosine residue to the COOH terminus of the detyrosinated form of alpha-tubulin. The measurement of hTTL mRNA expression in 74 primary neuroblastomas by quantitative real-time reverse transcription-PCR revealed that its high expression was significantly associated with favorable stages (1, 2 and 4s; p = 0.0069), high TrkA expression (p = 0.002), a single copy of MYCN (p < 0.00005), tumors found by mass screening (p = 0.0042), nonadrenal origin (p = 0.0042) and good prognosis (p = 0.023). The log-rank test showed that high expression of hTTL was an indicator of favorable prognosis (p = 0.026). Immunohistochemical analysis using specific antibodies generated by us demonstrated that tyrosinated tubulin (Tyr-tubulin), detyrosinated tubulin (Glu-tubulin) and hTTL as well as Delta2-tubulin were positive in favorable tumors, whereas only Delta2-tubulin was positive in the tumors with MYCN amplification. In an RTBM1 neuroblastoma cell line, hTTL was increased after treating the cells with bone morphogenetic protein 2 (BMP2) or all-trans retinoic acid (RA), which induced neuronal differentiation. These results suggest that the deregulated tubulin tyrosination/detyrosination cycle caused by decreased expression of hTTL is associated with inhibition of neuronal differentiation and enhancement of cell growth in the primary neuroblastomas with poor outcome.

Amino Acid Sequence↗

PubMed2003

Expression profiling and characterization of 4200 genes cloned from primary neuroblastomas: identification of 305 genes differentially expressed between favorable and unfavorable subsets.

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups: NBL in patients under 1 year of age usually regresses spontaneously, whereas that in patients over 1 year of age often grows aggressively and eventually kills the patient. To understand the molecular mechanism of biology and tumorigenesis of NBL, we decided to perform a comprehensive approach to unveil the gene expression profiles among the NBL subsets. We constructed the subset-specific oligo-capping cDNA libraries from the primary NBL tissues with favorable (F: stage 1, high expression of TrkA and a single copy of MYCN) and unfavorable (UF: stage 3 or 4, decreased expression of TrkA and MYCN amplification) characteristics and randomly cloned 4654 cDNAs. Among 4243 cDNAs sequenced successfully, 1799 (42.4%) were the genes with unknown function. Excluding the housekeeping genes, an expression profile of each subset was extremely different. To determine the genes expressed differentially between F and UF subsets, we performed semiquantitative reverse transcriptase (RT)-PCR for each of the 1842 independent genes using RNA obtained from 16 F and 16 UF NBLs as template. This revealed that 278 genes were highly expressed in the F subset as compared to the UF one, while, surprisingly, only 27 genes were expressed at higher levels in the UF rather than the F subset. These differentially expressed genes included 194 genes with unknown function. Many of the genes expressed at high levels in the F subset were related to catecholamine biosynthesis, small GTPases, synapse formation, synaptic vesicle transport, and transcription factors regulating differentiation of the neural crest-derived cells. On the other hand, the genes expressed at high levels in the UF subset included transcription factors and/or receptors that might regulate neuronal growth and differentiation. The chromosomal mapping of those genes showed some clusters. Thus, our mass-identification and characterization of the differentially expressed genes between the subsets may become a powerful tool for finding the important genes of NBL as well as developing new diagnostic and therapeutic strategies against aggressive NBL.

Chromosome Mapping↗

PubMed2003

Accumulation of p27 KIP1 is associated with BMP2-induced growth arrest and neuronal differentiation of human neuroblastoma-derived cell lines.

Bone morphogenetic proteins (BMPs) play an essential role in cell fate determination. In this study, we found that BMP2 treatment resulted in growth arrest and differentiation in human neuroblastoma-derived cell lines, SH-SY5Y and RTBM1. Within 30min of BMP2 exposure, phosphorylation of Smad1/5 was observed in these cell lines. In RTBM1 cells, BMP2-induced differentiation was accompanied by a significant decrease in the expression level of DAN, an antagonist of BMP in frog embryos. Immunoblot analysis revealed that BMP2 treatment caused a down-regulation of p53 family members and hence of cyclin-dependent kinase inhibitor p21(WAF1). We found a significant accumulation of p27(KIP1) in response to BMP2, whereas the expression level of Skp2, which is required for ubiquitin-dependent p27(KIP1) degradation, was decreased during this differentiation process. Our results suggest that p27(KIP1) contributes to the BMP-induced growth arrest and neuronal differentiation of neuroblastoma, and BMP treatment might provide a new therapeutic strategy.

Animals↗

PubMed2003

Neuroblastoma oligo-capping cDNA project: toward the understanding of the genesis and biology of neuroblastoma.

Neuroblastoma (NBL) is a common pediatric cancer originated from the neuronal precursor cells of sympathoadrenal lineage. NBLs show a variety of clinical phenotypes from spontaneous regression to malignant progression with acquirement of resistance to therapy. To understand the molecular mechanism of the genesis, progression, and regression of NBL, we need to identify key molecules determining the neuronal development of sympathoadrenal lineage. To this end, we have performed the NBL cDNA project. It includes (1) mass-cloning of the expressed genes from oligo-capping cDNA libraries derived from primary NBLs with different clinical and biological features; (2) mass-identification of differentially expressed genes between favorable and unfavorable subsets; and (3) molecular and functional analyses of the novel genes, which could be useful prognostic indicators. To date, 10,000 cDNA clones in total, approximately 40% of which contained novel sequences, were randomly picked up and DNA sequenced. We have identified approximately 500 differentially expressed genes between favorable and unfavorable subsets of NBL, among which more than 250 were the genes with unknown function.

DNA, Complementary↗

PubMed2003

Chromosomes that show partial loss or gain in near-diploid tumors coincide with chromosomes that show whole loss or gain in near-triploid tumors: evidence suggesting the involvement of the same genes in the tumorigenesis of high- and low-risk neuroblastomas.

We performed two-color fluorescence in situ hybridization analysis to detect the numbers of chromosomes 1 and 17, 1p deletion, and 17q gain in 177 neuroblastomas, including 101 tumors that were found by a mass-screening program for infants. Sixty-eight tumors with disomy 1 or tetrasomy 1 were classified as the Dis1 group, and 109 tumors with trisomy 1, pentasomy 1, or a mixed population of cells with trisomy 1 and cells with tetrasomy 1 were classified as the Tris1 group. 17q gain was the most frequent genetic event, followed by 1p deletion, and MYCN amplification in both Dis1 and Tris1 tumors. However, the incidence of all the genetic events was higher in Dis1 tumors than in Tris1 tumors. These findings suggest that Tris1 tumors are more resistant to acquiring the genetic events than are Dis1 tumors. In addition, there was an accumulation of genetic events in more advanced stages, with the exception of a high incidence of 17q gain in the stage IVS Tris1 tumors. Comparative genomic hybridization analysis, which was performed in 59 of the 177 tumors, showed that chromosomes partially lost or gained in Dis1 tumors coincided with chromosomes totally lost or gained in Tris1 tumors. Dis1 and Tris1 tumors were considered to have near-diploid/tetraploid and near-triploid/pentaploid chromosome numbers, respectively. These findings suggest that the same tumor-suppressor genes or oncogenes may be involved in the development and progression of both high- and low-risk neuroblastomas, and that the ploidy state of the tumor plays a fundamental role in the heterogeneous behavior.

Allelic Imbalance↗

PubMed2002

p73-dependent expression of DAN during cisplatin-induced cell death and osteoblast differentiation.

We previously reported that DAN, a founding member of the DAN family of secreted proteins, acts as an inhibitor of cell cycle progression and is closely involved in retinoic acid-induced neuroblastoma differentiation. In this study, we found that DAN as well as p73, the recently identified p53 family member, was up-regulated during osteoblast differentiation. Additionally, the expression of DAN was increased in response to cisplatin-induced cell death of neuroblastoma SH-SY5Y cells. Consistent with the previous reports, p73 was accumulated after the treatment with cisplatin. Intriguingly, we found a putative p53/p73-binding site in the 5'-upstream region of the human DAN gene. A luciferase reporter assay and an in vitro DNA-binding experiment revealed that this canonical p53/p73-binding site was a functional responsive element and was specific for p73. Our results suggest that there exists a functional association between DAN and p73 during osteoblast differentiation as well as cisplatin-induced cell death.

Animals↗