Masatoshi Kusuhara

Endothelin Potentiates Growth Factor‐stimulated DNA Synthesis in Swiss 3T3 Cells

A murine cell line, Swiss 3T3, is commonly used for the study of cellular growth. The present study revealed that this cell line possesses high‐affinity receptors for endothelin, a vasoactive peptide derived from vascular endothelial cells. In this cell line, endothelin potentiated DNA synthesis stimulated by platelet‐derived growth factor, basic fibroblast growth factor or insulin. The endothelin concentration required for potentiating DNA synthesis in this cell line is almost the same as that reported previously in endothelin‐induced vasoconstriction. Since endothelin increased intracellular calcium levels, this ion may participate in the signal transduction pathways by which endothelin induces potentiation of DNA synthesis.

Comparison of biological activities of endothelin-1, -2 and -3 in murine and human fibroblast cell lines

The effects of three forms of endothelin (ET), ET-1, -2 and -3, on intracellular free Ca2+ concentration ([Ca2+]i) and their receptor binding activities have been compared in murine fibroblast cell line Swiss 3T3 as well as diploid human fibroblast cell line FS-4. In both cell lines, ET-1 and -2 induced a profound increase in [Ca2+]i with a similar dose-response relationship; ET-3 was far less potent. ET-1 and -2 showed specific binding to both cell lines with similar dissociation constants and numbers of binding sites. Negligible specific binding of ET-3 was detected. These findings indicate that these two fibroblast populations possess high affinity receptors for ET-1 and -2 and practically no ET-3 receptors for ET-3, and binding of ET-1 and -2 increases [Ca2+]i in these cell lines.

Mobilization of intracellular calcium by endothelin in Swiss 3T3 cells

When intracellular free Ca2+ concentration [( Ca2+]i) was monitored in fura2-loaded Swiss 3T3 cells, endothelin increased [Ca2+]i in a dose-dependent manner; after the addition of endothelin, an initial transient peak was observed immediately and was followed by a sustained increase in [Ca2+]i lasting at least 5 min. 45Ca2+ efflux and influx experiments in endothelin-stimulated Swiss 3T3 cells revealed that the change in [Ca2+]i could be explained by a dual mechanism; an initial transient peak induced mainly by the release of Ca2+ from intracellular stores and the sustained increase by an influx of extracellular Ca2+. Cellular generation of inositol 1,4,5-trisphosphate and cyclic AMP were not induced by endothelin, suggesting that other cellular mediators with the capacity to release Ca2+ from intracellular stores play a significant role in the signal transduction pathway of endothelin in Swiss 3T3 cells.

Effect of endothelin as a coronary vasoconstrictor in the Langendorff-perfused rat heart

The effects of endothelin on coronary circulation were studied in isolated rat heart using a constant-flow system. Endothelin (10 fmol to 20 pmol/g heart weight) elevated the coronary perfusion pressure in a dose-dependent manner. Nifedipine (100 nM) inhibited the vasoconstriction and shifted the dose-response curve to the right. These results indicate that endothelin is a potent coronary vasoconstrictor and that its action is possibly mediated by the nifedipine-dependent Ca2+ channel.

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.

Atrial natriuretic peptide secretion in rabbits with aortic regurgitation

SummaryWe investigated whether or not the extent of secretion of atrial natriuretic peptide (ANP) was altered during the chronic course of aortic regurgitation (AR) in rabbits. AR was induced by aortic valve perforation in 20 rabbits. Left ventricular end-diastolic pressure (LVEDP) was used as an index of the severity of heart failure. LVEDP and plasma immunoreactive (IR)-ANP were measured before and at 15 min, 1 week, and 4 weeks after induction of AR. In each period a correlation between LVEDP and plasma IR-ANP was observed, and the coefficients and covariances did not vary throughout the experiment. We conclude, therefore, that a chronic change in ANP secretion does not develop during the first 4 weeks after induction of AR in rabbits.

Organotypic brain explant culture as a drug evaluation system for malignant brain tumors

Therapeutic options for malignant brain tumors are limited, with new drugs being continuously evaluated. Organotypic brain slice culture has been adopted for neuroscience studies as a system that preserves brain architecture, cellular function, and the vascular network. However, the suitability of brain explants for anticancer drug evaluation has been unclear. We here adopted a mouse model of malignant glioma based on expression of H‐RasV12 in Ink4a/Arf−/− neural stem/progenitor cells to establish tumor‐bearing brain explants from adult mice. We treated the slices with cisplatin, temozolomide, paclitaxel, or tranilast and investigated the minimal assays required to assess drug effects. Serial fluorescence‐based tumor imaging was sufficient for evaluation of cisplatin, a drug with a pronounced cytotoxic action, whereas immunostaining of cleaved caspase 3 (a marker of apoptosis) and of Ki67 (a marker of cell proliferation) was necessary for the assessment of temozolomide action and immunostaining for phosphorylated histone H3 (a marker of mitosis) allowed visualization of paclitaxel‐specific effects. Staining for cleaved caspase 3 was also informative in the assessment of drug toxicity for normal brain tissue. Incubation of explants with fluorescently labeled antibodies to CD31 allowed real‐time imaging of the microvascular network and complemented time‐lapse imaging of tumor cell invasion into surrounding tissue. Our results suggest that a combination of fluorescence imaging and immunohistological staining allows a unified assessment of the effects of various classes of drug on the survival, proliferation, and invasion of glioma cells, and that organotypic brain slice culture is therefore a useful tool for evaluation of antiglioma drugs.

CD44 variant 9 expression as a predictor for gastric cancer recurrence: immunohistochemical and metabolomic analysis of surgically resected tissues

CD44 variant 9 (CD44v9) and the heavy chain of 4F2 cell-surface antigen (CD98hc) appear important for regulation of reactive oxygen species defence and tumor growth in gastric cancer. This study examined the roles of CD44v9 and CD98hc as markers of gastric cancer recurrence, and investigated associations with energy metabolism. We applied capillary electrophoresis time-of-flight mass spectrometry to metabolome profiling of gastric cancer specimens from 103 patients who underwent resection with no residual tumor or microscopic residual tumor, and compared metabolite levels to immunohistochemical staining for CD44v9 and CD98hc. Positive expression rates were 40.7% for CD44v9 and 42.7% for CD98hc. Various tumor characteristics were significantly associated with CD44v9 expression. Five-year recurrence-free survival rate was significantly lower for CD44v9-positive tumors (39.1%) than for CD44v9-negative tumors (73.5%; P < 0.0001), but no significant differences in recurrence-free survival were seen according to CD98hc expression. Uni- and multivariate analyses identified positive CD44v9 expression as an independent predictor of poorer recurrence-free survival. Metabolome analysis of 110 metabolites found that levels of glutathione disulfide were significantly lower and reduced glutathione (GSH)/ glutathione disulfide (GSSG) ratio was significantly higher in CD44v9-positive tumors than in CD44v9-negative tumors, suggesting that CD44v9 may enhance pentose phosphate pathway flux and maintain GSH levels in cancer cells.

Prevalence of low-penetrant germline TP53 D49H mutation in Japanese cancer patients

Using whole exome sequencing data obtained from 1,685 Japanese cancer patients, we examined genetic variations of germline TP53 and found 10 types of non-synonymous single nucleotide variants. In the present study, we focused on 6 patients with germline D49H mutation located in the transactivation domain 2 of p53 protein, since the mutation seemed to be prevalent in cancer patients and to be pathogenic. According to the initial survey for family history of the proband with the germline TP53 D49H mutation, one osteosarcoma patient and his pedigree fulfill the criteria for Li-Fraumeni-like syndrome and the 2009 Chompret criteria for germline TP53 mutation screening. Since this patient possesses double germline mutations of TP53 D49H and A159D, further studies are required to evaluate contribution of the D49H mutation in this morbidity. The remaining 5 patients had family histories of cancer, but none fulfills the criteria either for the Li-Fraumeni/Li-Fraumeni-like syndromes or the 2009 Chompret criteria for germline TP53 mutation screening. It is possible to postulate that the germline TP53 D49H mutation is likely to be low-penetrant in some pedigrees. The present study also indicates that the survey for the germline TP53 mutation plays an important role in clinical practice as it will prevent mistaking cancer patients with unusual heredities for sporadic cases.