Biomedical subjects

Akira Nakagawara

Publications and source records attributed to Akira Nakagawara.

At least 19 recordsLinked to original sources

PubMed2007

Reciprocal expression of CCAAT/enhancer binding proteins alpha and beta in hepatoblastomas and its prognostic significance.

Hepatoblastoma is one of the common pediatric solid tumors with frequent mutation of the beta-catenin gene which might be an early event of its carcinogenesis. However, the detailed molecular mechanism is still unknown. We studied the expression levels of CCAAT/enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta, which regulate differentiation and growth of embryonic hepatocytes, to establish whether or not they were involved in affecting the clinical behavior of hepatoblastoma. The quantitative real-time reverse transcriptase-PCR revealed that expression of C/EBPalpha mRNA was significantly up-regulated in tumors 223% (p=0.013) as compared with that in adjacent normal livers, while expression of C/EBPbeta was down-regulated to 27% (p=0.002). Of interest, the immunohistochemical analysis showed that expression of C/EBPalpha was higher and that of C/EBPbeta lower in the poorly differentiated tumor cells than in the well-differentiated cells within the same tumor. Furthermore, high expression of C/EBPalpha (p=0.047) as well as low expression of C/EBPbeta (p=0.025) was significantly associated with poor prognosis of the patients. Cox hazard model suggested that expression of C/EBPalpha and that of C/EBPbeta were independent indicators to predict the prognosis from age but not from histology. Thus, expression of C/EBP proteins may play an important role in the genesis and clinical behavior of hepatoblastoma probably by inducing different stages of arrest of differentiation.

CCAAT-Enhancer-Binding Protein-alpha↗

PubMed2006

The intracellular domain of the amyloid precursor protein (AICD) enhances the p53-mediated apoptosis.

Amyloid precursor protein (APP)-derived intracellular domain (AICD) has a cytotoxic effect on neuronal cells and also participates in the regulation of gene transactivation. However, the precise molecular mechanisms behind the AICD-mediated apoptosis remain unknown. In this study, we have demonstrated that AICD interacts with p53 and enhances its transcriptional and pro-apoptotic functions. p53 was induced to be accumulated and associated with APP in response to cisplatin. Indeed, APP-C57 was co-immunoprecipitated with the endogenous p53. Enforced expression of APP-C57 or APP-C59 in U2OS cells bearing wild-type p53 led to an increase in number of apoptotic cells, whereas they had undetectable effects on p53-deficient H1299 cells, suggesting that AICD contributes to the activation of the p53-mediated apoptotic pathway. Consistent with this notion, the p53-mediated transcriptional activation and apoptosis were significantly enhanced by co-expression with APP-C57 or APP-C59. Thus, our present results strongly suggest that AICD triggers apoptosis through the p53-dependent mechanisms.

Amyloid beta-Protein Precursor↗

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

Critical role of PICT-1, a tumor suppressor candidate, in phosphatidylinositol 3,4,5-trisphosphate signals and tumorigenic transformation.

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) regulates diverse cellular functions by dephosphorylating the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)). Recent study revealed that PICT-1/GLTSCR2 bound to and stabilized PTEN protein in cells, implicating its roles in PTEN-governed PIP(3) signals. In this study, we demonstrate that RNA interference-mediated knockdown of PICT-1 in HeLa cells down-regulated endogenous PTEN and resulted in the activation of PIP(3) downstream effectors, such as protein kinase B/Akt. Furthermore, the PICT-1 knockdown promoted HeLa cell proliferation; however the proliferation of PTEN-null cells was not altered by the PICT-1 knockdown, suggesting its dependency on PTEN status. In addition, apoptosis of HeLa cells induced by staurosporine or serum-depletion was alleviated by the PICT-1 knockdown in the similar PTEN-dependent manner. Most strikingly, the PICT-1 knockdown in HeLa and NIH3T3 cells promoted anchorage-independent growth, a hallmark of tumorigenic transformation. Furthermore, PICT-1 was aberrantly expressed in 18 (41%) of 44 human neuroblastoma specimens, and the PICT-1 loss was associated with reduced PTEN protein expression in spite of the existence of PTEN mRNA. Collectively, these results suggest that PICT-1 plays a role in PIP(3) signals through controlling PTEN protein stability and the impairment in the PICT-1-PTEN regulatory unit may become a causative factor in human tumor(s).

Animals↗

PubMed2006

p73 and MDM2 confer the resistance of epidermoid carcinoma to cisplatin by blocking p53.

p73 responds to DNA damage and exerts its pro-apoptotic function. However, p73 might contribute to the development of drug-resistance in certain tumor cells. In this study, we found that p73 and MDM2 correlate with cisplatin-resistant phenotype of human epidermoid carcinoma-derived cells. p73 and MDM2 were kept at low levels in the cisplatin-sensitive KB-3-1 cells, whereas p53 was induced to be phosphorylated at Ser-15 in response to cisplatin. In contrast, p73 and MDM2 were expressed at higher levels, and cisplatin-mediated p53 phosphorylation was undetectable in the cisplatin-resistant KCP-4 cells. Enforced expression of p73 in KB-3-1 cells caused an accumulation of unphosphorylated form of p53 and MDM2, and conferred the cisplatin resistance. Collectively, our results suggest that a loss of the cisplatin sensitivity is at least in part due to a lack of cisplatin-induced p53 phosphorylation, and p73 might cooperate with MDM2 to be involved in this process.

Antineoplastic Agents↗

PubMed2006

p73-dependent induction of 14-3-3sigma increases the chemo-sensitivity of drug-resistant human breast cancers.

It has been well documented that tumor suppressor p53 is mutated in about 50% of all human tumors. p53 status might be one of the critical determinants for the chemo-sensitivity of human tumors. In the present study, we have found that p53 family member p73 as well as 14-3-3sigma is down-regulated in response to adriamycin (ADR) in ADR-resistant human breast cancer-derived MBA-MD-436 cells which carry p53 mutation. Like p53, 14-3-3sigma was transactivated by p73 and, in turn, stabilized p73. Luciferase reporter analysis and colony formation assays demonstrated that 14-3-3sigma has an ability to enhance the p73-mediated transcriptional activity as well as its pro-apoptotic function. Furthermore, enforced expression of 14-3-3sigma increased the ADR sensitivity of MBA-MD-436 cells. Taken together, our present results strongly suggest that p73-dependent induction of 14-3-3sigma plays an important role in the regulation of chemo-sensitivity of breast cancers bearing p53 mutation.

14-3-3 Proteins↗

PubMed2006

Marked and independent prognostic significance of the CpG island methylator phenotype in neuroblastomas.

The CpG island methylator phenotype (CIMP) was closely associated with poor overall survival (OS) in Japanese neuroblastoma (NBL) cases in our previous study. Here, in German NBL cases, CIMP(+) cases (n=95) showed markedly poorer OS (hazard ratio (HR)=9.5; P<0.0001) and disease-free survival (DFS) (HR=5.4; P<0.0001) than CIMP(-) cases (n=50). All the 23 cases with N-myc amplification had CIMP. Among the remaining cases without N-myc amplification, CIMP(+) cases (n=27) had a poorer OS (HR=4.5; P=0.02) and DFS (HR=5.2; P<0.0001) than CIMP(-) cases (n=95). In multivariate analysis, CIMP and N-myc amplification had an influence on OS and DFS independent of age and disease stage. CIMP had a stronger influence on DFS than N-myc amplification while N-myc had a stronger influence on OS.

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

Biology of neuroblastomas that were found by mass screening at 6 months of age in Japan.

BACKGROUND: Mass screening (MS) of neuroblastoma has been carried out by measuring the urinary catecholamine metabolites in infants at the age of 6 months in Japan. We assessed the incidence of neuroblastoma that may be a target for MS by studying tumor biology. PROCEDURE: FISH on chromosome 1 and MYCN analysis was performed on 453 patients that were classified into three clinical groups (287 infants found by MS, 51 infants < 12 months diagnosed clinically, and 115 children >or=12 months diagnosed clinically). The relationship between the biological types of tumors and the clinical outcome was examined. RESULTS: Type 1 (trisomy 1 and normal MYCN), type 2 (disomy 1/tetrasomy 1 and normal MYCN), and type 3 (disomy 1/tetrasomy 1 and amplified MYCN) tumors were found in 88.2%, 10.5%, and 1.4% of infants found by MS, in 68.0%, 24.0%, and 8.0% of infants diagnosed clinically, and in 23.4%, 42.3%, and 34.2% of children diagnosed clinically (P < 0.001). Infants with type 1 tumors found by MS or diagnosed clinically had earlier stages of the disease (P < 0.0001 and P = 0.0005) and better overall survival (P < 0.001 and P = 0.005) than children with type 1 tumors diagnosed clinically. Infants with type 2 tumors found by MS, had earlier stages (P = 0.06 and P < 0.0001) and better overall survival (P = 0.014 and P < 0.001) than infants or children with type 2 tumors diagnosed clinically. All three clinical groups of patients with type 3 tumors had advanced stages and dismal prognoses. CONCLUSIONS: About 12% of tumors found by MS showed unfavorable biological (types 2 and 3) characteristics.

Adolescent↗

PubMed2006

Overlapping roles for homeodomain-interacting protein kinases hipk1 and hipk2 in the mediation of cell growth in response to morphogenetic and genotoxic signals.

Homeodomain-interacting protein kinase 1 (Hipk1), 2, and 3 genes encode evolutionarily conserved nuclear serine/threonine kinases, which were originally identified as interacting with homeodomain-containing proteins. Hipks have been repeatedly identified as interactors for a vast range of functional proteins, including not only transcriptional regulators and chromatin modifiers but also cytoplasmic signal transducers, transmembrane proteins, and the E2 component of SUMO ligase. Gain-of-function experiments using cultured cells indicate growth regulatory roles for Hipks on receipt of morphogenetic and genotoxic signals. However, Hipk1 and Hipk2 singly deficient mice were grossly normal, and this is expected to be due to a functional redundancy between Hipk1 and Hipk2. Therefore, we addressed the physiological roles of Hipk family proteins by using Hipk1 Hipk2 double mutants. Hipk1 Hipk2 double homozygotes are progressively lost between 9.5 and 12.5 days postcoitus and frequently fail to close the anterior neuropore and exhibit exencephaly. This is most likely due to defective proliferation in the neural fold and underlying paraxial mesoderm, particularly in the ventral region, which may be attributed to decreased responsiveness to Sonic hedgehog signals. The present study indicated the overlapping roles for Hipk1 and Hipk2 in mediating cell proliferation and apoptosis in response to morphogenetic and genotoxic signals during mouse development.

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

UFD2a mediates the proteasomal turnover of p73 without promoting p73 ubiquitination.

p73 protein level is kept extremely low in mammalian cultured cells and its stability may be regulated by not only the ubiquitin/proteasome-dependent proteolysis but also through other unidentified mechanisms. Here, we found for the first time that p73 is physically as well as functionally associated with the U-box-type E3/E4 ubiquitin ligase UFD2a. The immunoprecipitation experiments demonstrated that this interaction is mediated by the COOH-terminal region of p73alpha containing SAM domain. During the cisplatin-induced apoptosis in SH-SY5Y neuroblastoma cells, p73alpha accumulated at a protein level, whereas the endogenous UFD2a was significantly reduced in response to cisplatin. Ectopic expression of UFD2a decreased the half-life of p73alpha in association with a significant inhibition of the p73alpha-mediated transactivation as well as proapoptotic activity. Downregulation of endogenous UFD2a by antisense strategy resulted in a remarkable accumulation of p73alpha. Unexpectedly, UFD2a-mediated degradation of p73alpha was sensitive to the proteasomal inhibitor, however, UFD2a did not affect the ubiquitination levels of p73alpha. Taken together, our present findings imply that UFD2a might promote the proteasomal turnover of p73 in a ubiquitination-independent manner, and also suggest that UFD2a might play an important role in the regulation of cisplatin-induced apoptosis mediated by p73.

Animals↗

PubMed2005

Functional implication of p73 protein stability in neuronal cell survival and death.

p73, a newly identified member of p53 family, locates at human chromosome 1p36.2-3, a region which is frequently deleted in a wide variety of human tumors including neuroblastoma. p73 is induced to be accumulated in response to a subset of DNA damaging agents such as cisplatin, and thereby promoting G1/S cell cycle arrest and/or apoptosis. Since the expression levels of p73 are kept extremely low under normal conditions, stabilization of p73 is critical for its effects on cell growth inhibition and apoptosis. Indeed, p73 is induced at protein level in SH-SY5Y neuroblastoma cells exposed to cisplatin. Several lines of evidence indicate that stress-induced post-translational modifications of p73 such as phosphorylation and acetylation lead to a marked extension of its half-life. p73 stability is regulated at least in part by proteasome-dependent degradation pathway, however, MDM2 which mediates ubiquitination and subsequent degradation of p53 by the 26S proteasome, does not promote the proteolytic degradation of p73, implying that the protein stability of p73 is regulated through a pathway distinct from that of p53. Although little is known about the regulation of p73 turnover, we are now beginning to understand the regulatory mechanisms by which p73 is induced to be stabilized in response to apoptotic stimuli, and exerts its pro-apoptotic activity. In this review, we discuss about the cellular proteins implicated in the stability control of p73.

Cell Death↗

PubMed2005

A review of DNA microarray analysis of human neuroblastomas.

Neuroblastoma (NBL) is an enigmatic tumor with heterogeneous clinical behaviors including maturation, regression, and aggressive growth. Despite recent progress in therapeutic strategies against advanced NBL, long-term outcomes still remain very poor. The prediction of cancer prognosis is one of the most urgent demands to initiate the suitable treatment of NBL. Recent papers have demonstrated that cancers can be diagnosed on the basis of gene expression profiling. We have been proceeded NBL cDNA project to collect a large number of genes expressed in NBLs, to identify the genes differentially expressed between favorable and unfavorable NBLs, and to make an NBL-proper cDNA chip for large-scale analysis of NBL tumors. Computational analysis of gene expression data in NBLs identified many prognosis-related genes and provided a classifier to predict the patient prognosis with high efficiency. Conversion of these findings into better diagnosis and treatment is now underway. Thus, molecular profiling of NBL has become a feasible tool for clinical applications.

DNA, Complementary↗

PubMed2005

Prediction of MYCN amplification in neuroblastoma using serum DNA and real-time quantitative polymerase chain reaction.

PURPOSE: MYCN amplification (MNA) indicates a poor prognosis in neuroblastoma (NB) and is routinely assayed for therapy stratification. We aimed to develop a diagnostic tool to predict MYCN status using serum DNA, which, in cancer patients, predominantly originates from tumor-released DNA. PATIENTS AND METHODS: Using DNA-based real-time quantitative polymerase chain reaction, we simultaneously quantified MYCN (2p24) and a reference gene, NAGK (2p12), and evaluated MYCN copy number as an MYCN/NAGK (M/N) ratio in 87 NB patients whose MYCN status had been determined by Southern blotting. Of these patients, 17 had MYCN-amplified NB, and 70 had nonamplified NB. RESULTS: The serum M/N ratio in the MNA group (median, 199.32; range, 17.1 to 901.6; 99% CI, 107.0 to 528.7) was significantly (P < .001) higher than the ratio in the non-MNA group (median, 0.87; range, 0.25 to 4.6; 99% CI, 0.82 to 1.26; Mann-Whitney U test). The sensitivity and specificity of the serum M/N ratio as a diagnostic test were both 100% when the serum M/N ratio cutoff was set at 10.0. Among six MNA patients whose clinical courses were followed, the serum ratios decreased to the normal range in the patients in remission (n = 3), whereas the ratios increased to high levels in the patients who relapsed (n = 2) or failed to achieve remission (n = 1). CONCLUSION: Measurement of the serum M/N ratio seems to be a promising method for accurately assessing MYCN status in NB, although a larger set of patients needs to be examined to confirm this result.

Adolescent↗

PubMed2005

LMO3 interacts with neuronal transcription factor, HEN2, and acts as an oncogene in neuroblastoma.

LIM-only proteins (LMO), which consist of LMO1, LMO2, LMO3, and LMO4, are involved in cell fate determination and differentiation during embryonic development. Accumulating evidence suggests that LMO1 and LMO2 act as oncogenic proteins in T-cell acute lymphoblastic leukemia, whereas LMO4 has recently been implicated in the genesis of breast cancer. However, little is known about the role of LMO3 in either tumorigenesis or development. In the present study, we have identified LMO3 and HEN2, which encodes a neuronal basic helix-loop-helix protein, as genes whose expression levels were higher in unfavorable neuroblastomas compared with those of favorable tumors. Immunoprecipitation and immunostaining experiments showed that LMO3 was associated with HEN2 in mammalian cell nucleus. Human neuroblastoma SH-SY5Y cells stably overexpressing LMO3 showed a marked increase in cell growth, a promotion of colony formation in soft agar medium, and a rapid tumor growth in nude mice compared with the control transfectants. More importantly, the increased expression of LMO3 and HEN2 was significantly associated with a poor prognosis in 87 primary neuroblastomas. These results suggest that the deregulated expression of neuronal-specific LMO3 and HEN2 contributes to the genesis and progression of human neuroblastoma in a lineage-specific manner.

Adaptor Proteins, Signal Transducing↗

PubMed2005

topors, a p53 and topoisomerase I-binding RING finger protein, is a coactivator of p53 in growth suppression induced by DNA damage.

The RING family zinc-finger protein topors (topoisomerase I-binding protein) binds not only topoisomerase I, but also p53 and the AAV-2 Rep78/68 proteins. topors maps to human chromosome 9p21, which contains candidate tumor suppressor genes implicated in small cell lung cancers. In this study, we isolated the murine counterpart of topors and investigated its impact on p53 function. The deduced amino-acid sequence of mouse topors exhibits extensive similarity to human topors. Overexpressed myc-tagged topors associates with and stabilizes p53, and enhances the p53-dependent transcriptional activities of p21(Waf1), MDM2 and Bax promoters and elevates endogenous p21(Waf1) mRNA levels. Overexpression of topors consequently results in the suppression of cell growth by cell cycle arrest and/or by the induction of apoptosis. Taken together, these studies identify topors as a positive regulator of p53. The expression of topors is induced by exposure to the genotoxic reagents cisplatin and camptothecin, a DNA topoisomerase I inhibitor. We therefore postulate that topors mediates p53-dependent cellular responses induced by DNA damage, suggesting its physiological role as a tumor suppressor.

Amino Acid Sequence↗

PubMed2005

Identification of protein kinase A catalytic subunit beta as a novel binding partner of p73 and regulation of p73 function.

Post-translational modifications play a crucial role in regulation of the protein stability and pro-apoptotic function of p53 as well as its close relative p73. Using a yeast two-hybrid screening based on the Sos recruitment system, we identified protein kinase A catalytic subunit beta (PKA-Cbeta) as a novel binding partner of p73. Co-immunoprecipitation and glutathione S-transferase pull-down assays revealed that p73alpha associated with PKA-Cbeta in mammalian cells and that their interaction was mediated by both the N- and C-terminal regions of p73alpha. In contrast, p53 failed to bind to PKA-Cbeta. In vitro phosphorylation assay demonstrated that glutathione S-transferase-p73alpha-(1-130), which has one putative PKA phosphorylation site, was phosphorylated by PKA. Enforced expression of PKA-Cbeta resulted in significant inhibition of the transactivation function and pro-apoptotic activity of p73alpha, whereas a kinase-deficient mutant of PKA-Cbeta had no detectable effect. Consistent with this notion, treatment with H-89 (an ATP analog that functions as a PKA inhibitor) reversed the dibutyryl cAMP-mediated inhibition of p73alpha. Of particular interest, PKA-Cbeta facilitated the intramolecular interaction of p73alpha, thereby masking the N-terminal transactivation domain with the C-terminal inhibitory domain. Thus, our findings indicate a PKA-Cbeta-mediated inhibitory mechanism of p73 function.

Animals↗