Kenichi Urakami

Enzyme‐linked Immunosorbent Assay of Pro‐gastrin‐releasing Peptide for Small Cell Lung Cancer Patients in Comparison with Neuron‐specific Enolase Measurement

Our previous study demonstrated that pro‐gastrin‐releasing peptide(31–98), or ProGRP, is a specific tumor marker in patients with small cell lung carcinoma (SCLC). Using a newly developed, highly sensitive enzyme‐linked immunosorbent assay (ELISA) for ProGRP, we analyzed 1,446 samples including those obtained from 478 lung cancer patients to evaluate the clinical usefulness of this ELISA. Several properties indicated that ProGRP is a useful tumor marker for SCLC. First, ProGRP was specifically elevated in SCLC patients. In non‐SCLC patients and patients with non‐tumorous lung diseases, its serum level was very rarely elevated. Secondly, ProGRP was a reliable marker, in terms of the marked elevation of serum ProGRP levels in SCLC patients. Thirdly, serum ProGRP levels were elevated in SCLC patients even at a relatively early stage of this disease. Fourthly, changes in the serum ProGRP level showed an excellent correlation with the therapeutic responses in SCLC patients. Neuron‐specific enolase (NSE) is accepted as a tumor marker of SCLC patients. With the aim of comparing ProGRP and NSE as tumor markers for SCLC patients, we measured serum NSE levels in all samples collected in the present study. We found that ProGRP was superior to NSE in terms of sensitivity, specificity and reliability. Therefore, we consider that ProGRP can play a major role as a clinical tumor marker for SCLC patients.

Invitro and invivo antagonists against parathyroid hormone-related protein

Four analogues of parathyroid hormone-related protein (PTHrP), PTHrP(7-34)NH2, (10-34)NH2, (15-34)NH2 and (20-34)NH2, were synthesized and their antagonistic activity against PTHrP(1-34) was examined in vitro and in vivo. In vitro studies revealed that all four analogues antagonized PTHrP-stimulated cyclic AMP production in rat osteosarcoma cells (ROS 17/2.8), and that PTHrP(7-34)NH2 and PTHrP(10-34)NH2 had potent antagonistic activity. In vivo experiments in nude mice also revealed that PTHrP(7-34)NH2 completely inhibited hypercalcemia induced by PTHrP(1-34), indicating that these analogues antagonize the effects of PTHrP(1-34) in vitro and in vivo.

Serial analysis of matrix metalloproteinase-2 in dialysate of rat sclerosing peritonitis models

AbstractBackground. Sclerosing peritonitis (SP) is a serious complication of continuous ambulatory peritoneal dialysis (CAPD). In order to carry out CAPD safely, it is important to analyze the development of SP. Methods. We prepared animal models of SP by the intraperitoneal administration of chlorhexidine gluconate (CHX) or talc, using male Sprague-Dawley rats. The peritoneal equilibration test and histological examinations were performed in the model rats, and dialysate drained from them was analyzed by gelatin zymography. Results. In the two types of SP animal models, matrix metalloproteinase-2 (MMP-2) level in dialysate correlated with the changes of inflammation, thickness of peritoneum, D/D0 glucose level (glucose level of drained dialysate obtained 90 min after the injection of 2.5% glucose containing peritoneal dialysis fluid divided by that obtained immediately after the injection), and net ultrafiltration. Conclusions. From these results, MMP-2 level in drained dialysate was considered to change with the development and progression of SP in rat models. Thus, MMP-2 has potential as a diagnostic marker for SP.

Implementation of individualized medicine for cancer patients by multiomics-based analyses—the Project HOPE—

The Project HOPE (High-tech Omics-based Patient Evaluation) for cancer medicine aims to evaluate biological characteristics of each cancer tissue as well as diathesis of each patient in around 1,000 consecutive cases per year, who receive operations at the Shizuoka Cancer Center. Cancer tissues are investigated by whole-exome sequencing for 18,835 genes, focusing on 12,776 in-house cancer hotspots from 483 cancer-associated genes. To confirm cancer-specific genetic changes, we analyzed blood cells to collate with data of cancer tissues, and we reevaluate cancer tissues by comprehensive cancer panel for 409 genes. In order to investigate diathesis of the patients, we evaluate 43,015 hotspots associated with non-cancerous diseases. In terms of gene expression profiling, we analyze cancer-specific alterations for 29,833 genes using tumor and adjacent normal tissues. If and when necessary, we investigate tumor and normal tissues by proteomics and metabolomics. The model experiments using glioblastoma cell lines demonstrated that the method is appropriate for clinical application. The Project HOPE makes it possible to implement individualized medicine and to practice preventive and presymptomatic medicine for cancer patients. Furthermore, the project can create important seeds for research and development in cancer medicine.

Inhibitory Effect of 8‐Chloro‐cyclic Adenosine 3′,5′‐Monophosphate on Cell Growth of Gastric Carcinoma Cell Lines

A cAMP analogue, 8‐chloro‐cAMP (8‐Cl‐cAMP), selectively binds to site 1 receptor of type II regulatory subunit (RII) of cAMP‐dependent protein kinase. The effects of 8‐Cl‐cAMP on human gastric carcinoma cell lines were studied. Twenty μM 8‐CI‐cAMP clearly inhibited cell growth in six cell lines (TMK‐1, KATO‐III, MKN‐7, ‐28, ‐45, and ‐74) but not in MKN‐1. Cell population in the G1 phase was increased in KATO III cells, which were most responsive to 8‐Cl‐cAMP, while cell cycle progression in TMK‐1 and MKN‐1 cells was apparently not influenced by 8‐Cl‐cAMP. The various changes induced by 8‐Cl‐cAMP were further analyzed in TMK‐1 cells. Decrease of type I regulatory subunit (RI) of cAMP‐dependent protein kinase and translocation of RII from cytosol to nucleus were induced by 8‐Cl‐cAMP treatment. 8‐CI‐cAMP increased the level of cAMP‐response element (CRE) binding protein in addition to inducing FOS mRNA, whose promoter contains CRE. 8‐Cl‐cAMP decreased the expression of mRNA for transforming growth factor‐α (TGF‐α), while the expression of epidermal growth factor receptor was not changed. Expression of HRAS and MYC mRNAs was slightly increased, whereas the amounts of HRAS and MYC proteins remained unchanged. Our results overall suggest that 8‐Cl‐cAMP might be a useful tool for antitnmor therapy of gastric cancers and that cell growth inhibition by 8‐Cl‐cAMP might account for the decrease of TGF‐α expression by tumor cells.

Next generation sequencing approach for detecting 491 fusion genes from human cancer.

Next-generation DNA sequencing (NGS) of the genomes of cancer cells is contributing to new discoveries that illuminate the mechanisms of tumorigenesis. To this end, the International Cancer Genome Consortium and The Cancer Genome Atlas are investigating novel alterations of genes that will define the pathways and mechanisms of the development and growth of cancers. These efforts contribute to the development of innovative pharmaceuticals as well as to the introduction of genome sequencing as a component of personalized medicine. In particular, chromosomal translocations that fuse coding sequences serve as important pharmaceutical targets and diagnostic markers given their association with tumorigenesis. Although increasing numbers of fusion genes are being discovered using NGS, the methodology used to identify such fusion genes is complicated, expensive, and requires relatively large samples. Here, to address these problems, we describe the design and development of a panel of 491 fusion genes that performed well in the analysis of cultured human cancer cell lines and 600 clinical tumor specimens.

Integrated analysis of gene expression and copy number identified potential cancer driver genes with amplification-dependent overexpression in 1,454 solid tumors

Identification of driver genes contributes to the understanding of cancer etiology and is imperative for the development of individualized therapies. Gene amplification is a major event in oncogenesis. Driver genes with tumor-specific amplification-dependent overexpression can be therapeutic targets. In this study, we aimed to identify amplification-dependent driver genes in 1,454 solid tumors, across more than 15 cancer types, by integrative analysis of gene expression and copy number. Amplification-dependent overexpression of 64 known driver oncogenes were found in 587 tumors (40%); genes frequently observed were MYC (25%) and MET (18%) in colorectal cancer; SKP2 (21%) in lung squamous cell carcinoma; HIST1H3B (19%) and MYCN (13%) in liver cancer; KIT (57%) in gastrointestinal stromal tumors; and FOXL2 (12%) in squamous cell carcinoma across tissues. Genomic aberrations in 138 known cancer driver genes and 491 established fusion genes were found in 1,127 tumors (78%). Further analyses of 820 cancer-related genes revealed 16 as potential driver genes, with amplification-dependent overexpression restricted to the remaining 22% of samples (327 tumors) initially undetermined genetic drivers. Among them, AXL, which encodes a receptor tyrosine kinase, was recurrently overexpressed and amplified in sarcomas. Our studies of amplification-dependent overexpression identified potential drug targets in individual tumors.

Integrated next-generation sequencing analysis of whole exome and 409 cancer-related genes

The use of next-generation sequencing (NGS) techniques to analyze the genomes of cancer cells has identified numerous genomic alterations, including single-base substitutions, small insertions and deletions, amplification, recombination, and epigenetic modifications. NGS contributes to the clinical management of patients as well as new discoveries that identify the mechanisms of tumorigenesis. Moreover, analysis of gene panels targeting actionable mutations enhances efforts to optimize the selection of chemotherapeutic regimens. However, whole genome sequencing takes several days and costs at least

Optimizing an ion semiconductor sequencing data analysis method to identify somatic mutations in the genomes of cancer cells in clinical tissue samples

10,000, depending on sequence coverage. Therefore, laboratories with relatively limited resources must employ a more economical approach. For this purpose, we conducted an integrated nucleotide sequence analysis of a panel of 409-cancer related genes (409-CRG) combined with whole exome sequencing (WES). Analysis of the 409-CRG panel detected low-frequency variants with high sensitivity, and WES identified moderate and high frequency somatic variants as well as germline variants.

Gene-mediated expression and partial characterization of ATPase 7B in cultured cells

Identification of causal genomic alterations is an indispensable step in the implementation of personalized cancer medicine. Analytical methods play a central role in identifying such changes because of the vast amount of data produced by next generation sequencer. Most analytical techniques are designed for the Illumina platform and are therefore suboptimal for analyzing datasets generated by whole exome sequencing (WES) using the Ion Proton System. Accurate identification of somatic mutations requires the characterization of platform-dependent error profiles and genomic properties that affect the accuracy of sequence data as well as platform-oriented optimization of the pipeline. Therefore, we used the Ion Proton System to perform WES of DNAs isolated from tumor and matched control tissues of 1,058 patients with cancer who were treated at the Shizuoka Cancer Center Hospital. Among the initially identified candidate somatic single-nucleotide variants (SNVs), 10,279 were validated by manual inspection of the WES data followed by Sanger sequencing. These validated SNVs were used as an objective standard to determine an optimum cutoff value to improve the pipeline. Using this optimized pipeline analysis, 189,381 SNVs were identified in 1,101 samples. The analytical technique presented here is a useful resource for conducting clinical WES, particularly using semiconductor-based sequencing technology.