Kazuya Omi

Long-lasting RNAi activity in mammalian neurons

The effect of RNA interference (RNAi) induced by synthetic small interfering RNAs (siRNAs) on proliferating mammalian cells appears to last for approximately 3–7 days after its induction. Here we show that the RNAi activity induced by a synthetic 21‐nucleotide siRNA duplex in postmitotic neurons, mouse primary hippocampal neurons and neurons that differentiated from mouse embryonal carcinoma P19 cells persists for at least 3 weeks, suggesting long‐lasting RNAi activity in mammalian neurons. In addition, we also show that an apoptotic (or antiviral) pathway triggered by long dsRNAs is generated during neuronal differentiation of P19 cells, by which the sequence‐specific RNAi activity involving long dsRNA appears to be masked.

CD36 polymorphism is associated with protection from cerebral malaria.

The human protein CD36 is a major receptor for Plasmodium falciparum-infected erythrocytes and contributes to the pathology of P. falciparum malaria. We performed variation screening of the CD36 gene and examined the possible association between CD36 polymorphisms and the severity of malaria in 475 adult Thai patients with P. falciparum malaria. Accordingly, we identified nine CD36 polymorphisms with a high-frequency (>15%) minor allele. Of these, the frequencies of the -14T-->C allele in the upstream promoter region and the -53G-->T allele in the downstream promoter region were significantly decreased in patients with cerebral malaria compared to those with mild malaria (P=.016 for -14T-->C and P=.050 for -53G-->T). The analysis of linkage disequilibrium (LD) between the nine common polymorphisms revealed that there are two blocks with strong LD in the CD36 gene and that the -14T-->C and -53G-->T polymorphisms are within the upstream block of 35 kb from the upstream promoter to exon 8. Further association testing after the second variation screening in the upstream block indicated that the in3(TG)(12) (i.e., 12 TG repeats in intron 3) allele is most strongly associated with the reduction in the risk of cerebral malaria (odds ratio 0.59; 95% confidence interval 0.40-0.87; P=.0069). We found, by reverse-transcriptase PCR amplification, that in3(TG)(12) is involved in the nonproduction of the variant CD36 transcript that lacks exons 4 and 5. Since exon 5 of the gene is known to encode the ligand-binding domain for P. falciparum-infected erythrocytes, in3(TG)(12) itself or a primary variant on the haplotype with in3(TG)(12) may be responsible for protection from cerebral malaria in Thailand. Results of the present study suggest that LD mapping has potential for detecting a disease-associated variant on the basis of haplotype blocks.

Fcγ receptor IIA and IIIB polymorphisms are associated with susceptibility to cerebral malaria

Human FcgammaRIIA and FcgammaRIIIB exhibit genetic polymorphisms, FcgammaRIIA-131H/R and FcgammaRIIIB-NA1/NA2, coding for different capacities for IgG binding and phagocytosis. Recently, FcgammaRIIA-131R was reported to be associated with protection against high-density Plasmodium falciparum infection in Kenya. Furthermore, FcgammaRIIIB-NA1/NA2 polymorphism was shown to influence FcgammaRIIA function in an allele-specific manner. In this study, we examined a possible association of FcgammaRIIA-131H/R and FcgammaRIIIB-NA1/NA2 polymorphisms with malaria severity in 107 cerebral malaria patients, 157 non-cerebral severe malaria patients, and 202 mild malaria controls living in northwest Thailand. This study reveals that, with the FcgammaRIIIB-NA2 allele, the FcgammaRIIA-131H/H genotype is associated with susceptibility to cerebral malaria (OR 1.85, 95% CI 1.14-3.01; P=0.012), although these polymorphisms are not individually involved in the disease severity. Our results suggest that FcgammaRIIA-131H/R and FcgammaRIIIB-NA1/NA2 polymorphisms have an interactive effect on host defense against malaria infection.

14-3-3zeta is indispensable for aggregate formation of polyglutamine-expanded huntingtin protein.

Huntingtons disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by polyglutamine (polyQ) expansions in the huntingtin (Htt) protein. A hallmark of HD is the presence of aggregates-predominantly composed of NH(2)-terminal fragments of polyQ-expanded Htt-in the nucleus and cytoplasm of affected neurons. We previously proposed that 14-3-3zeta might act as a sweeper of misfolded proteins by facilitating the formation of aggregates possibly for neuroprotection; these aggregates are referred to as inclusion bodies. However, evidence available in this regard is indirect and circumstantial. In this study, analysis of the aggregation-prone protein Htt encoded by HD gene exon 1 containing polyglutamine expansions (Htt86Q) revealed that 17 residues in the NH(2)-terminal of this protein are indispensable for its aggregate formation. Immunoprecipitation assays revealed that 14-3-3beta, gamma, eta, and zeta interact with Htt86Q transfected in N2a cells. Interestingly, the small interfering ribonucleic acid (siRNA) suppression of 14-3-3zeta exclusively abolished Htt86Q aggregate formation, whereas 14-3-3beta or eta siRNA suppression did not. This indicates that 14-3-3zeta participates in aggregate formation under nonnative conditions. Our data support a novel role for 14-3-3zeta in the aggregate formation of nonnative, aggregation-prone proteins.

Cisplatin influences acquisition of resistance to molecular-targeted agents through epithelial-mesenchymal transition-like changes.

Chemotherapy with platinum agents is the standard of care for non‐small‐cell lung cancer (NSCLC); however, novel molecular‐targeted agents like gefitinib have been approved for advanced NSCLCs, including recurrent cases previously treated with platinum‐based chemotherapy. Although these agents show antitumor activity through distinct mechanisms and elicit positive initial responses, tumors invariably develop resistance. Recent studies have revealed mechanisms by which both types of agents induce acquired resistance. However, little is known about whether first‐line treatment with either type of agent affects cancer cell susceptibility and development of resistance against subsequent treatment with the other. Using in vitro drug‐resistant NSCLC cell models, we provide evidence that acquired cisplatin resistance may reduce the sensitivity of cancer cells to subsequent treatment with a molecular‐targeted agent. In addition, first‐line cisplatin treatment influenced the mechanism by which cancer cells developed resistance to subsequent treatment with a molecular‐targeted agent. The influence of cisplatin on acquisition of resistance to a molecular‐targeted agent was associated with epithelial–mesenchymal transition (EMT)‐like alterations such as increased expression of mesenchymal markers, morphological change, and AXL tyrosine kinase‐mediated increased cell motility. Our findings indicate that the influence of platinum‐based chemotherapy on molecular‐targeted therapies and the involvement of EMT and EMT‐related effectors should be considered when developing therapeutic strategies using antitumor agents, especially in the context of sequential therapy.

Noncompetitive Immunoassay Detection System for Haptens on the Basis of Antimetatype Antibodies

BACKGROUND Small molecules classified as haptens are generally measured by competitive immunoassay, which is theoretically inferior to noncompetitive sandwich immunoassay in terms of sensitivity and specificity. We created a method for developing sandwich immunoassays to measure haptens on the basis of antimetatype antibodies. METHODS We generated antimetatype monoclonal antibodies against a hapten-antibody immunocomplex using an ex vivo antibody development system, the Autonomously Diversifying Library (ADLib) system. We selected 2 haptens, estradiol (E2) and 25-hydroxyvitamin D [25(OH)D], as analytes. Sandwich immunoassays for these 2 haptens were developed by use of a 96-well microtiter plate and a fully automated chemiluminescence analyzer, and the performances of these immunoassays were investigated. RESULTS The developed assays exhibited sensitivity high enough to detect target haptens in serum samples. The limit of detection of the ELISA for E2 was 3.13 pg/mL, and that of the fully automated chemiluminescent enzyme immunoassay (CLEIA) system was 2.1 ng/mL for 25(OH)D. The cross-reactivity with immunoreactive derivatives was effectively improved compared with the competitive assay. The CVs for the sandwich ELISA for E2 were 4.2%-12.6% (intraassay) and 6.2%-21.8% (total imprecision). The CVs for the sandwich CLEIA for 25(OH)D were 1.0%-2.3% (intraassay) and 1.9%-3.5% (total imprecision). In particular, the sandwich CLEIA for 25(OH)D showed correlations of r = 0.99 with both LC-MS/MS and a commercially available (125)I RIA. CONCLUSIONS Our method represents a potentially simple and practical approach for routine assays of haptens, including vitamins, hormones, drugs, and toxins.

Protein kinase D2 and heat shock protein 90 beta are required for BCL6-associated zinc finger protein mRNA stabilization induced by vascular endothelial growth factor-A

Vascular endothelial growth factor (VEGF) is a major angiogenic factor that activates pro-angiogenic molecules to generate new vessels. Recently, we identified a VEGF-A-induced pro-angiogenic gene, BCL-6 associated zinc finger protein (BAZF), in endothelial cells. BAZF interacts with CBF1, a transcriptional regulator of Notch signaling, and downregulates Notch signaling by inducing the degradation of CBF1. A signal inhibition assay with a combination of chemical inhibitors and siRNA revealed that the protein kinase D (PRKD) family, mainly PRKD2, mediated BAZF gene expression by VEGF-A stimulation. A luciferase reporter assay showed that the promoter activity of the BAZF gene was unchanged by VEGF-A stimulation. However, we found that the stability of BAZF mRNA increased in a VEGF-A/PRKD2-dependent manner. In further studies to investigate the underlying mechanism, we successfully identified heat shock protein 90 beta (HSP90β) as a molecule that interacts with and stabilizes BAZF mRNA following VEGF-A/PRKD2 activation. These data suggest that HSP90β may positively regulate angiogenesis, not only as a protein chaperone, but also as an mRNA stabilizer for pro-angiogenic genes, such as BAZF, in a PRKD2 activity-dependent manner.

Novel monoclonal antibodies recognizing the active conformation of epidermal growth factor receptor.

The precise regulation of epidermal growth factor receptor (EGFR) is crucial for its function in cellular growth control. Although many antibodies against EGFR have been developed and used to analyze its regulation and function, it is not yet easy to analyze activated EGFR specifically. Activated EGFR has been mainly detected by its phosphorylation state using anti-phospho-EGFR and anti-phosphotyrosine antibodies. In the present study, we have established novel monoclonal antibodies which recognize the activated EGFR independently of its phosphorylation. Our antibodies detected active state of EGFR in immunoprecipitation and immunofluorescence, by recognizing the epitopes which are exposed through the conformational change induced by ligand-binding. Furthermore, we found that our antibodies preferentially detected the conformation of constitutively active EGFR mutants found in lung cancer cell lines. These results indicate that our antibodies may become novel research and diagnostic tools for detecting and analyzing the conformation of active EGFR in various cells and tissues.

Association study of the chemokine, CXC motif, ligand 1 (CXCL1) gene with sporadic Alzheimer's disease in a Japanese population

Inflammation is profoundly involved in the development of Alzheimers disease (AD) and other neurodegenerative diseases. Chemokine, CXC motif, ligand 1 (CXCL1; or GRO1) is an inflammatory cytokine and appears to be implicated in the pathogenesis of AD. It is of interest and importance to see if the CXCL1 gene, mapped on chromosome 4q12-q13, has potential for conferring the predisposition to AD. Here we report on an association study of the CXCL1 gene with sporadic AD patients in a Japanese population; three single nucleotide polymorphisms (SNPs) in the CXCL1 locus were investigated in 103 AD patients and 130 healthy individuals. The results indicate that neither genotype frequencies nor allele frequencies of the examined SNPs attained statistical significance even after being stratified by the presence or absence of the Apolipoprotein E epsilon4 allele. Therefore, the data presented here suggests that the CXCL1 gene could not be associated with the susceptibility to AD in a Japanese population.

Abstract B46: Soluble AXL is a candidate circulating biomarker for predicting acquired and intrinsic resistance to molecular-targeted agents in NSCLC cells.

Background: Drug resistance is an important factor that compromise therapeutic effect of most anti-cancer drugs including molecular-targeted agents. Non-invasive assessment of drug resistance by circulating biomarkers would therefore be useful for determining therapeutic options for resistant patients. Recently, a receptor tyrosine kinase AXL has been implicated in acquired resistance to EGFR inhibitors in NSCLC cells. In this study, we examined the potential utility of AXL as a circulating biomarker to predict acquired or intrinsic drug resistance using NSCLC cells. Materials and methods: Drug resistant NSCLC cell lines were developed by exposing NSCLC cells with either EGFR inhibitor gefitinib or MET inhibitor PHA-665752. AXL-positive cells in parent and established resistant cell lines were isolated by anti-AXL antibody-immobilized magnetic beads. Drug resistance was assessed by MTS growth inhibition assay. Immunoblot analysis was performed on cell lysate and conditioned medium to evaluate expression and release of AXL. The role of AXL in cell growth and motility was examined by MTS growth inhibition assay and transwell migration assay. Results: Drug-resistant NSCLC cell lines were successfully established, and increased release of fragmented soluble form of AXL was observed in these cell lines. They showed increased cell motility, which was effectively inhibited by AXL knockdown using siRNA. In contrast, their cell growth was not affected by AXL knockdown, even when these cells were simultaneously treated with molecular-targeted agents. Furthermore, cells highly expressing AXL were immunologically isolated from a gefitinib-sensitive parent cell line. These cells showed partial resistance to gefitinib, indicating that such cells intrinsically exist in NSCLC cell populations. Conclusions: Soluble form of AXL is a candidate circulating biomarker for predicting acquired and intrinsic resistance to molecular-targeted agents in NSCLC cells. It may also be used to predict AXL-mediated increased motility of resistant cells. Patients harboring such resistant cells may have increased chance of responding to AXL-targeted anti-metastasis therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B46. Citation Format: Nobuyuki Ise, Nobuaki Takemori, Kazuya Omi, Katsutoshi Goishi, Shigeki Higashiyama. Soluble AXL is a candidate circulating biomarker for predicting acquired and intrinsic resistance to molecular-targeted agents in NSCLC cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B46.