Masaki Takiguchi

Autologous antibody to src-homology 3-domain GRB2-like 1 specifically increases in the sera of patients with low-grade gliomas

BackgroundGlioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable in spite of various therapeutic approaches. Clarification of the oncogenic process in its early stage is important for the diagnosis and effective therapy.MethodsIn the present study, we used the serological identification of antigens by recombinant cDNA expression cloning (SEREX) to explore the subtle changes of the protein expression in low-grade glioma. The levels of serum autoantibodies to the SEREX-identified glioma-related antigens were analyzed by ELISA, and the epitope site was identified using deletion mutants and overlap peptide array. Changes in the serum autoantibody levels were examined in the rat glioma model using C6 and 9u2009L glioma cell lines.ResultsWe identified 31 glioma-related antigens by SEREX. Among them, the serum level of autoantibody to src-homology 3-domain GRB2-like 1 (SH3GL1) was significantly higher in patients with low-grade glioma than healthy volunteers or high-grade gliomas. The 10 amino-acids at the C-terminal were identified as the epitope site by the overlap peptide array and the ELISA using deletion mutants. The tissue expression of SH3GL1 protein increased in proportion to glioma progression. The rat glioma models confirmed the increase of anti-SH3GL1 autoantibody level in the early stage and the suppression in the late stage.ConclusionSH3GL1 may be involved in the oncogenic process of gliomas and effectively elicit an autologous antibody response in low-grade gliomas. The immunological reaction to SH3GL1 would contribute to the establishment of a novel diagnostic and therapeutic target for gliomas.

Identification of a novel SEREX antigen family, ECSA, in esophageal squamous cell carcinoma

BackgroundDiagnosis of esophageal squamous cell carcinoma (SCC) may improve with early diagnosis. Currently it is difficult to diagnose SCC in the early stage because there is a limited number of tumor markers available.ResultsFifty-two esophageal SCC SEREX antigens were identified by SEREX (serological identification of antigens by recombinant cDNA expression cloning) using a cDNA phage library and sera of patients with esophageal SCC. Sequence analysis revealed that three of these antigens were similar in amino acid sequences, and they were designated as ECSA (e sophageal c arcinoma S EREX a ntigen)-1, -2 and -3. The ECSA family was also similar to an EST clone, hepatocellular carcinoma-associated antigen 25a (HCA25a). Serum antibody levels to ECSA-1, -2 and -3 were significantly higher in patients with esophageal SCC than in healthy donors. Based on the conserved amino acid sequences, three peptides were synthesized and used for enzyme-linked immunosorbent assays (ELISA). The serum antibody levels against one of these peptides were significantly higher in patients with esophageal SCC. This peptide sequence was also conserved in FAM119A, GOSR1 and BBS5, suggesting that these are also ECSA family members. Reverse transcription followed by quantitative PCR analysis showed that the mRNA expression levels of ECSA-1, -2 and -3 and FAM119A but not of HCA25a, GOSR1 and BBS5 were frequently elevated in esophageal SCC tissues.ConclusionsWe have identified a new gene family designated ECSA. Serum antibodies against the conserved domain of the ECSA family may be a promising tumor marker for esophageal SCC.

Calpain regulates thymidylate synthase–5‐fluoro‐dUMP complex levels associated with response to 5‐fluorouracil in gastric cancer cells

Thymidylate synthase (TS) plays a major role in the response to 5‐fluorouracil (5‐FU) by binding directly to the 5‐FU metabolite, 5‐fluoro‐dUMP (FdUMP). The change in the TS expression levels after 5‐FU administration was examined in parallel to 5‐FU responsiveness in six human gastric adenocarcinoma cell lines to elucidate the source of variability of 5‐FU sensitivity. MKN‐1, SH‐10‐TC and MKN‐74 cells were more resistant to 5‐FU than MKN‐28, KATO III and MKN‐45 cells. Western blotting analysis revealed that the 5‐FU sensitivity of these cells did not correlate with the basal TS expression levels but did correlate with rapid detection of the TS‐FdUMP complex after exposure to 5‐FU. In 5‐FU‐resistant cells, very low levels of the TS‐FdUMP complex early after 5‐FU exposure were elevated by pretreatment with calpain inhibitors such as benzyloxycarbonyl‐leucyl‐leucinal (ZLLH), benzyloxycarbonyl‐leucyl‐leucyl‐leucinal (ZLLLH) and ALLN, but not by other protease inhibitors. In contrast, ONO‐3403, which causes calpain activation, stimulated downregulation of the TS‐FdUMP complex in 5‐FU‐sensitive cells. The expression levels of calpastatin, an endogenous calpain inhibitor, were higher in 5‐FU‐sensitive cells than in 5‐FU‐resistant cells. ZLLH increased the 5‐FU sensitivity of 5‐FU‐resistant cells, whereas ONO‐3403 decreased the sensitivity of 5‐FU‐sensitive cells. In addition, knockdown of m‐calpain by siRNA increased the 5‐FU sensitivity in 5‐FU‐resistant cells, while knockdown of calpastatin reduced the sensitivity in 5‐FU‐sensitive cells. These results suggest that calpain might reduce the chemosensitivity of human gastric cancer cells to 5‐FU possibly by rapid degradation of the TS‐FdUMP complex, a finding that is considered to have novel therapeutic implications. (Cancer Sci 2011; 102: 1509–1515)

Identification of stroke-associated-antigens via screening of recombinant proteins from the human expression cDNA library (SEREX)

BackgroundBecause circulating antibodies against a variety of antigens have been detected in patients with coronary heart disease, carotid atherosclerosis and those who have suffered a stroke, it is suspected that immune response may be one of the mechanisms of atherogenesis The objective of this study is to identify novel antibodies in ischemic stroke patients by screening the expressed recombinant proteins using a human cDNA library (SEREX).MethodsTo identify the candidate antigens, cDNA library was screened by SEREX using plasma from ten patients with ischemic stroke. Subsequently, via ELISA using recombinant proteins and synthetic peptides, the serum antibody levels were measured in two independent patient/healthy donor (HD) cohorts (142 and 78 in the 2nd screening and a validation cohort, respectively).ResultsThe initial screening resulted in the identification of six candidate antigens. Of these antigens, replication protein A2 (RPA2) was determined to be the antigen associated with stroke (Pu2009<u20090.05) by ELISA with 2nd screening and validation cohort. Multifactorial logistic regression analysis showed that the increased levels of the RPA2 antibodies (RPA2-Abs) associated with stroke independent of other risk factors for stroke (Pu2009<u20090.05). Receiver operating curve analysis demonstrated that the area under the curve from ELISA using GST fusion RPA2 and synthetic peptides (bRPA2-132) were 0.867 (95% CI: 0.798-0.936) and 0.971 (95% CI: 0.940-1.00), respectively. If the cut-off value of the bRPA2-132-Ab level was determined to be 0.334, the sensitivity and specificity of the antibody level as the diagnostic marker for stroke were 0.323 (95% CI: 0.209-0.453) and 1.00 (95% CI: 0.713-1.00), respectively.ConclusionsSEREX identified RPA2 as the antigen associated with ischemic stroke and serum auto-antibodies against RPA2 elevates in stroke patients. RPA2-Abs could become a biomarker for the evaluation of ischemic stroke at risk.

Elevated Serum Antibody Levels against Cyclin L2 in Patients with Esophageal Squamous Cell Carcinoma

Cyclin L2 (CCNL2) (Acc. No.: NM_030937) was detected as a tumor antigen of esophageal squamous cell carcinoma (SCC) by serological identification of antigens by recombinant cDNA expression cloning (SEREX). Serum anti-CCNL2 antibodies were detected by enzyme-linked immunosorbent assay more frequent in patients with esophageal SCC than in healthy donors (32% and 15%, P<0.01). An AlphaLISA further confirmed the significant difference in serum antibody levels between the patients and healthy donors using a different set of serum specimens. The expression levels of CCNL2 mRNA detected by reverse transcription polymerase chain reaction were higher in esophageal SCC tissues than those detected in adjacent normal esophageal tissues. We then analyzed for biological function by the transient transfection of CCNL2 expression plasmids into ras-NIH3T3 mouse fibroblasts followed by analysis via luciferase assay using p53-responsive reporter plasmids. CCNL2 increased the transactivation ability of p53, which was attenuated by protein kinase C (PKC) inhibitors or a dominant negative PKCa. Thus, it is possible that CCNL2 activates p53 via PKCa activation.

The secretogranin II gene is a signal integrator of glutamate and dopamine inputs

Cooperative gene regulation by different neurotransmitters likely underlies the long‐term forms of associative learning and memory, but this mechanism largely remains to be elucidated. Following cDNA microarray analysis for genes regulated by Ca2+ or cAMP, we found that the secretogranin II gene (Scg2) was cooperatively activated by glutamate and dopamine in primary cultured mouse hippocampal neurons. The Ca2+ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid acetoxymethyl ester (BAPTA‐AM) and the mitogen‐activated protein kinase (MAPK) kinase (MEK) inhibitor PD98059 prevented Scg2 activation by glutamate or dopamine; thus, the Ca2+/MEK pathway is predicted to include a convergence point(s) of glutamatergic and dopaminergic signaling. Unexpectedly, the protein kinase A inhibitor KT5720 enhanced Scg2 activation by dopamine. The protein‐synthesis inhibitor cycloheximide also enhanced Scg2 activation, and the proteasome inhibitor ZLLLH diminished the KT5720‐mediated augmentation of Scg2 activation. These results are concordant with the notion that dopaminergic input leads to accumulation of a KT5720‐sensitive transcriptional repressor, which is short‐lived because of rapid degradation by proteasomes. This repression pathway may effectively limit the time window permissive to Scg2 activation by in‐phase glutamate and dopamine inputs via the Ca2+/MEK pathway. We propose that the regulatory system of Scg2 expression is equipped with machinery that is refined for the signal integration of in‐phase synaptic inputs.

Functional Similarity of Anticancer Drugs by MTT Bioassay

We prepared normal or Ha-ras-transformed NIH3T3 cells transfected stably or transiently with various tumorrelated genes. The chemosensitivity of the transfected clones to 16 anticancer drugs was compared to the parental control cells using the MTT assay. The chemosensitivity changes induced by transfected genes were calculated and expressed numerically as the Drug Chemosensitivity Index (DCI). High DCI values (indicating resistance) were frequently observed in cells expressing C/EBP?, C/EBP?, p53, p21, PTEN, dominant-negative MDM2, caspases, HSP90, COUP-TF1 and decorin. In contrast, transfectants expressing ras, src, erbB2 and calpastatin had low DCI values, indicating increased sensitivity. Thus, it may be possible to predict the sensitivity of cancer cells toward anticancer drugs based on the expression levels of these genes. We then performed a regression analysis of DCI values between anticancer drugs. The correlation coefficients (r) were relatively high between cisplatin, camptothecin, mitomycin C and etoposide, suggesting that the mechanisms of action of these drugs are similar. The r values of aclarubicin, vincristine, taxol and cytarabine were low, suggesting that each of these drugs has a different and unique effect. This analysis may provide a rationale for design of combination chemotherapy regimens.

Novel serum autoantibodies against talin1 in multiple sclerosis: Possible pathogenetic roles of the antibodies

In the pathogenesis of multiple sclerosis (MS), B cell/antibody-related mechanisms have recently received attention. To investigate the role of autoantibody in MS, we performed SEREX which can identify autoantibody cyclopedically. We identified serum antibodies against cytoskeletal protein talin1, and the levels of whom were remarkably higher in 39 MS than 43 normal controls (P < 0.01) and 35 disease controls (P = 0.06), and in MS patients without oligoclonal bands than ones with them. Moreover, we found negative-correlations between serum anti-talin1 antibody and IgG index in MS (P = 0.03). Anti-talin1 antibody exists in MS patients sera, which may have some protective factor.

Circulating anti-filamin C autoantibody as a potential serum biomarker for low-grade gliomas

BackgroundGlioma is the most common primary malignant central nervous system tumor in adult, and is usually not curable due to its invasive nature. Establishment of serum biomarkers for glioma would be beneficial both for early diagnosis and adequate therapeutic intervention. Filamins are an actin cross-linker and filamin C (FLNC), normally restricted in muscle tissues, offers many signaling molecules an essential communication fields. Recently, filamins have been considered important for tumorigenesis in cancers.MethodsWe searched for novel glioma-associated antigens by serological identification of antigens utilizing recombinant cDNA expression cloning (SEREX), and found FLNC as a candidate protein. Tissue expressions of FLNC (both in normal and tumor tissues) were examined by immunohistochemistry and quantitative RT-PCR analyses. Serum anti-FLNC autoantibody level was measured by ELISA in normal volunteers and in the patients with various grade gliomas.ResultsFLNC was expressed in glioma tissues and its level got higher as tumor grade advanced. Anti-FLNC autoantibody was also detected in the serum of glioma patients, but its levels were inversely correlated with the tissue expression. Serum anti-FLNC autoantibody level was significantly higher in low-grade glioma patients than in high-grade glioma patients or in normal volunteers, which was confirmed in an independent validation set of patients’ sera. The autoantibody levels in the patients with meningioma or cerebral infarction were at the same level of normal volunteers, and they were significantly lower than that of low-grade gliomas. Total IgG and anti-glutatione S-transferase (GST) antibody level were not altered among the patient groups, which suggest that the autoantibody response was specific for FLNC.ConclusionsThe present results suggest that serum anti-FLNC autoantibody can be a potential serum biomarker for early diagnosis of low-grade gliomas while it needs a large-scale clinical study.

Identification of the p53-Responsive Element in the Promoter Region of the Human Decorin Gene

We have obtained some evidence that shows that the decorin gene is the target of p53 transactivation. Luciferase reporter plasmid, which contained the promoter region between positions -252 and -205, was activated by p53 dose- dependently up to 170-fold. The promoter region involved a sequence, 5’-AGGCAAGTAG-3’, similar to p53-binding consensus sequence, 5’-PuPuPuC(A/T)(A/T)GPyPyPy-3’. Chromatin immunoprecipitation assay using p53 antibodies revealed that the region between -413 and -232 of the promoter of the decorin gene was co-precipitated with p53. p53- binding to this region was further demonstrated by electrophoretic mobility shift assay, in which the complex between decorin promoter DNA and proteins decreased by pretreatment with anti-p53 antibodies. The mRNA expression levels of decorin increased after treatment with p53-activating nutlin-3 greatly and with genotoxic reagent, adriamycin, to some extent. Consequently, decorin promoter is useful to evaluate the p53 transactivation ability.