Tetsuya Ueda

HMGB proteins and transcriptional regulation.

In eukaryotic nuclei, transcription reactions on the chromatin are regulated by the binding of transcription regulators such as transcription factors and chromatin remodeling factors to chromatin. High-mobility-group-box (HMGB) protein family is a member of HMG super family proteins, the most abundant and ubiquitous non-histone chromatin binding proteins in eukaryotic cells. HMGB proteins bind to chromosomal DNA via their DNA binding motif, a HMG box, and induce structural changes of chromatin. Besides the chromatin binding property of HMGB proteins, HMGB proteins also interact with other proteins including transcription regulators and histones. In addition to those key transcriptional regulatory proteins, undoubtedly HMGB proteins bind dynamically to chromatin and interact with other proteins including transcription factors, thereby participating in transcription regulation in multiple processes. We will focus on the transcription regulation by HMGB proteins bound to chromatin, and possible roles of the unique structural and functional domain, the acidic C-tail region.

Distinct domains in HMGB1 are involved in specific intramolecular and nucleosomal interactions.

HMGB1 is composed of two DNA-binding domains and a long acidic tail at the C-terminus. The acidic tail interacts with the DNA-binding domains of HMGB1 and with core histone H3 in the nucleosome. These interactions are important for modulation of the DNA and chromatin binding activities of HMGB1, as well as biological functions of HMGB1. However, the interactions are not fully characterized, because the tertiary structure of full-length HMGB1 is still unknown. Here we use chemical cross-linking, mass spectrometry, and epitope masking analysis to perform a detailed characterization of the inter- and intramolecular protein interactions of the acidic tail of HMGB1. We show that specific regions of the acidic tail participate in intramolecular interactions with Lys2 of HMGB1 and in intermolecular interactions with Lys36 and Lys37 of histone H3. The acidic tail is oriented by its location adjacent to the C-terminus of helix III of DNA-binding HMG box A in the HMGB1 molecule. These results suggest that the acidic tail modulates the biological functions of HMGB1 through these specific interactions.

Detection of Minimal Gastric Cancer Cells in Peritoneal Washings by Focused Microarray Analysis with Multiple Markers: Clinical Implications

BackgroundPeritoneal cytology is an important prognostic factor of gastric cancer. However, peritoneal cytology requires great skill, which may explain its low prevalence. A reverse transcriptase–polymerase chain reaction–based assay with multiple marker genes or immunocytochemistry was assessed as an alternative method of gathering the same kind of data as cytology.MethodsPeritoneal washings from 179 patients with gastric cancer were analyzed by multiplex reverse transcriptase–polymerase chain reaction with 10 marker genes and subsequent hybridization to a customized oligo-nucleotide array. Results with this assay were either validated as a prognostic factor or confirmed by demonstrating the presence of cancer cells by immunocytochemical cytology.ResultsOnly 1 (2.2%) of 44 disease-free cases was shown to be positive by the microarray assay, whereas 13 (93%) of 14 conventional cytology–positive cases were found to be positive. This assay further detected approximately one-third of cytology-negative patients either with peritoneal recurrence (7 of 20, 35%) or with non-peritoneal recurrence (6 of 22, 27%). A high concordance between the microarray assay and immunocytochemical cytology with five antibodies against CK20, FABP1, MUC2, TFF1, and MASPIN was confirmed. The clinical outcome of the microarray assay–positive cases was poor, as was that of the cytology-positive cases.ConclusionsOur assay, though time-consuming and requiring special equipment, demonstrated a specificity and sensitivity equal to or better than cytology in our institutes. The minimal free peritoneal cancer cells detected by the microarray assay may provide the same clinical information as larger amounts of cancer cells for patients with gastric cancer. An anti-MASPIN antibody may be helpful in peritoneal cytology of gastric cancer.

Development of spectral colour banding in cytogenetic analysis

We developed a novel chromosome banding technique-spectral colour banding (SCAN). With this technique we displayed a multicolour banding pattern that almost entirely correlated with the corresponding G-banding pattern. With SCAN analysis we could identify the chromosome-band origin of double minute chromosomes in gastric cancer. Our preliminary use of this technique suggests that it has significant clinical applications for cytogenetic analysis.

Efficacy of Triple Therapy Plus Cetraxate for the Helicobacter pylori Eradication in Partial Gastrectomy Patients

In the present study, we aimed to establish an additional standardized protocol with a higher H. pylori eradication rate in the remnant stomach. Fifty-five H. pylori–positive patients were randomly allocated to one of three regimens: LAC—lansoprazole, amoxicillin, and clarithromycin b.i.d. for 7 days (n = 17); LAC+CET—LAC b.i.d. plus cetraxate q.i.d. for 7 days (n = 20); and LEFT—LAC for 7 days in a horizontal body position on the left side for 30 min (n = 18). Patient compliance and side effects were checked via interviews. H. pylori eradication was successful in 75, 72, and 41% in LAC+CET, LEFT, and LAC, respectively. The eradication rate was significantly higher in LAC+CET than in LAC (P = 0.024) but not in LEFT (P = 0.058). Adverse events that occurred in each group were almost all mild ones. Cetraxate plus LAC for 1 week is a safe and effective regime for the eradication of H. pylori in patients after partial gastrectomy.

Involvement of HMGB1 and HMGB2 proteins in exogenous DNA integration reaction into the genome of HeLa S3 cells

High mobility group 1 and 2 proteins (HMGB1 and HMGB2) are abundant chromosomal proteins in eukaryotic cells. We examined the involvement of HMGB1 and HMGB2 in nonhomologous illegitimate recombination. The HMGB1 or HMGB2 expression plasmid, carrying the neo(r) gene as a selection marker, was introduced into HeLa S3 cells to obtain stably-transfected cells. The number of G418-resistant colonies was about 10 times the number of colonies of control cells transfected with plasmids not carrying the HMGB genes. The copy number of the stably-integrated neo(r) gene was higher in the cells transfected with the HMGB expression plasmids than in control cells. The exogenous DNA integration was suggested to have occurred by nonhomologous illegitimate recombination. On the contrary, the introduction of the HMGB antisense RNA expression plasmid with a reporter plasmid carrying the neo(r) gene into HeLa S3 cells decreased the number of G418-resistant colonies. These results indicate that HMGB1 and HMGB2 each have a novel function as stimulators of stable integration of plasmid DNA into the host genome and that they may be important for the process of spontaneous DNA integration in living cells.

Effect of imatinib mesylate in a patient with a metastatic gastrointestinal stromal tumor with a c-kit mutation in exon 11.

A gastrointestinal stromal tumor (GIST) is a group of mesenchymal neoplasms that have been defined as KIT (CD117) or CD34 immunostaining-positive mesenchymal spindle cells or epithelioid neoplasms originating from the gastrointestinal tract [1–3]. Up to 90% of GISTs harbor a mutation in c-kit leading to KIT receptor autophosphorylation and ligand-independent activation, and such mutations appear to play a central role in the pathogenesis of GISTs [3, 4]. The c-kit mutations in GISTs are found most commonly in exon 11 and exon 9, but only rarely in exons coding for the kinase domain. The clinical significance and response to imatinib by the different types of mutation are not yet certain [4, 5]. Although surgical resection with curative intent is the accepted approach to patients presenting with localized GISTs, many such patients ultimately relapse, and about one half of GISTs are overtly metastatic at presentation, with the most common sites of metastases being the peritoneum and the