Toru Yashiro

Expression of mRNA for PACAP and its receptors in intra- and extra-adrenal human pheochromocytomas and their relationship to catecholamine synthesis.

PURPOSE Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagons/vasoactive intestinal peptide family, induces the expression of catecholamine-synthesizing enzymes in adrenal medullary cells. In addition, PACAP and its receptor have been detected in human pheochromocytoma tissues, though it is not yet known whether PACAP enhances the expression of genes encoding catecholamine-synthesizing enzymes. To address this question, we analyzed PACAP, PACAP receptor, and tyrosine hydroxylase (TH) and phenylethanolamine-N-methyltransferase (PNMT) mRNAs in pheochromocytomas. METHODS The levels of the mRNA for PACAP and vasoactive intestinal peptide (VIP), and their receptors, and for TH and PNMT were measured by RT-PCR or real-time PCR analysis, and the concentrations of catecholamines were measured by HPLC in 24 intra-adrenal and six extra-adrenal pheochromocytomas. RESULTS mRNA expression of PACAP and its receptor VPAC1R were detected in many pheochromocytomas (24/30 and 29/30, respectively), but mRNA expression of the PAC1R and VPAC2R receptor subtypes were detected in only one of six extra-adrenal pheochromocytomas. PACAP mRNA expression correlated with TH (p=0.0018) and PNMT (p=0.05) mRNA expression, as well as epinephrine (p=0.0342) levels in 16 intra-adrenal pheochromocytomas. CONCLUSION Our findings support a possible role for PACAP in the regulation of expression of genes encoding catecholamine-synthesizing enzymes in intra-adrenal pheochromocytomas.

Novel Germline Mutations in the SDHB and SDHD Genes in Japanese Pheochromocytomas

The SDHA, SDHB, SDHC, and SDHD genes code for subunits of succinate dehydrogenase (SDH), which forms part of the mitochondrial respiratory chain. Germline mutations in the genes encoding SDHB and SDHD have been reported in familial paragangliomas/pheochromocytomas and in apparently sporadic pheochromocytomas. SDHB and SDHD mutations are widely distributed along the genes with no apparent hot spots. SDHB mutations are often detected in malignant and extra-adrenal pheochromocytomas. SDHD mutations are also detected frequently in head and neck paragangliomas. We sequenced the entire coding regions of the SDHB and SDHD genes in 17 pheochromocytomas. Weidentified novel heterozygous G to A point mutations at the first base of intron 3 of the SDHB gene in a malignant extra-adrenal abdominal pheochromocytoma patient, and at the first base of codon 111 of the SDHD gene in an adrenal pheochromocytoma patient. Further, we confirmed the SDHD mutation by DHPLC. The prevalence of SDHB and SDHD mutations in pheochromocytomas we examined was 12% (2/17). Thus, we identified two novel SDH mutations in Japanese pheochromocytomas. Further studies will investigate the oncogenic potential of these mutations.

Development of High‐Speed Fluorescent X‐Ray Micro‐Computed Tomography

A high‐speed fluorescent x‐ray CT (FXCT) system using monochromatic synchrotron x rays was developed to detect very low concentration of medium‐Z elements for biomedical use. The system is equipped two types of high purity germanium detectors, and fast electronics and software. Preliminary images of a 10mm diameter plastic phantom containing channels field with iodine solutions of different concentrations showed a minimum detection level of 0.002 mg I/ml at an in‐plane spatial resolution of 100μm. Furthermore, the acquisition time was reduced about 1/2 comparing to previous system. The results indicate that FXCT is a highly sensitive imaging modality capable of detecting very low concentration of iodine, and that the method has potential in biomedical applications.

Human thyroid specimen imaging by fluorescent x-ray computed tomography with synchrotron radiation

Fluorescent x-ray computed tomography (FXCT) is being developed to detect non-radioactive contrast materials in living specimens. The FXCT system consists of a silicon (111) channel cut monochromator, an x-ray slit and a collimator for fluorescent x ray detection, a scanning table for the target organ and an x-ray detector for fluorescent x-ray and transmission x-ray. To reduce Compton scattering overlapped on the fluorescent K(alpha) line, incident monochromatic x-ray was set at 37 keV. The FXCT clearly imaged a human thyroid gland and iodine content was estimated quantitatively. In a case of hyperthyroidism, the two-dimensional distribution of iodine content was not uniform, and thyroid cancer had a small amount of iodine. FXCT can be used to detect iodine within thyroid gland quantitatively and to delineate its distribution.

Medical imaging by fluorescent x-ray CT: its preliminary clinical evaluation

Fluorescent x-ray CT (FXCT) with synchrotron radiation (SR) is being developed to detect the very low concentration of specific elements. The endogenous iodine of the human thyroid and the non-radioactive iodine labeled BMIPP in myocardium were imaged by FXCT. FXCT system consists of a silicon (111) double crystal monochromator, an x-ray slit, a scanning table for object positioning, a fluorescent x-ray detector, and a transmission x-ray detector. Monochromatic x-ray with 37 keV energy was collimated into a pencil beam (from 1 mm to 0.025 mm). FXCT clearly imaged endogenous iodine of thyroid and iodine labeled BMIPP in myocardium, whereas transmission x-ray CT could not demonstrate iodine. The distribution of iodine was heterogeneous within thyroid cancer, and its concentration was lower than that of normal thyroid. Distribution of BMIPP in normal rat myocardium was almost homogeneous; however, reduced uptake was slightly shown in ischemic region. FXCT is a highly sensitive imaging modality to detect very low concentration of specific element and will be applied to reveal endogenous iodine distribution in thyroid and to use tracer study with various kinds of labeled material.

Expression of mRNAs for succinate dehydrogenase subunits and related genes in pheochromocytoma.

Abstract:  Mutations in the genes encoding succinate dehydrogenase (SDH) have been associated with susceptibility to pheochromocytoma. However, few reports have examined the level of SDH mRNAs expression. In this study, we examined the level of expression of mRNAs encoding SDHB, SDHC, and SDHD in pheochromocytoma, pheochromocytoma subgroups, and normal adrenal gland, and compared the expression of these genes to the level of expression of related genes in the same tissues. The mean relative level of expression of SDHB, SDHC, SDHD and VHL mRNA was 28.7 ± 6.2%, 16.6 ± 4.8%, 214 ± 47.5% and 25.9 ± 8.2%, respectively, in pheochromocytoma tissues compared to normal adrenal gland. Furthermore, the mean relative level of the RET proto‐oncogene mRNA was 707 ± 149% in pheochromocytoma compared to normal adrenal gland. The level of expression of the SDH genes was highly correlated in each individual sample (P < 0.0001). The level of expression of the SDH mRNAs correlated with the level of VHL mRNA (P < 0.0001), but not with the level of RET mRNA. The level of SDH mRNAs expression also correlated with the expression of phenylethanolamine N‐methyl transferase (PNMT), an adrenaline synthesizing enzyme (P < 0.01), which may explain the correlation between SDH expression and adrenaline content (P < 0.05). The level of SDH mRNAs expression correlated strongly with the expression of VEGF mRNA (P < 0.0001). In multiple endocrine neoplasia (MEN) 2a, the expression of the SDH genes and VHL mRNA was significantly higher than that observed in adrenal or extra‐adrenal pheochromocytoma. The expression of the corticotropin‐releasing hormone (CRH) mRNA was significantly higher in extra‐adrenal pheochromocytoma than in adrenal pheochromocytoma or MEN2a. Thus, tumor‐specific gene expression exists in pheochromocytoma, which may explain the characteristics of the tumor.