Heiwa Okuda

Downregulation of Cap43 gene by von Hippel‐Lindau tumor suppressor protein in human renal cancer cells

We previously identified 9 genes (i.e., thymosin β4, secreted protein acidic and rich in cysteine, Cap43, ceruloplasmin, serum amyloid A, heat shock protein 90, LOT1, osteopontin and casein kinase Iγ) that are more highly expressed in cancerous regions than in noncancerous regions in human renal cancers. In our study, we considered the possibility that the von Hippel‐Lindau (VHL) tumor suppressor gene might be able to affect the expression of these 9 genes in renal cancer cells. We first established 2 VHL‐positive cell lines, 786/VHL‐1 and 786/VHL‐2, after the introduction of wild‐type VHL into VHL‐negative renal cancer 786‐O cells. Of these 9 genes, expression of the Cap43 gene was specifically downregulated by VHL. Expression of Cap43 was also much lower in 4 other VHL‐positive renal cancer cell lines than in VHL‐negative 786‐O cells. Cap43 promoter assays with several deletion or mutation constructs demonstrated that the Sp1 site in the element from –286 base pairs (bp) to –62 bp was partly responsible for VHL‐induced suppression of the Cap43 gene. Immunostaining analysis with human specimens of renal cancers demonstrated that the Cap43 protein was expressed in most cancer cells and macrophages. We also observed a marked and specific increase of Cap43 mRNA levels in response to hypoxia or nickel in all VHL‐positive cell lines. Cellular expression of Cap43 mRNA in response to hypoxia or nickel thus is closely associated with VHL gene expression in renal cancer cells. Although the function of the Cap43 protein remains unclear, the expression of Cap43 protein could be a molecular marker closely associated with VHL in renal cancer.

Epigenetic inactivation of the candidate tumor suppressor gene HOXB13 in human renal cell carcinoma.

Epigenetic alterations like DNA methylation and the resulting inactivation of cancer-related genes often contribute to the development of various cancers. To identify the genes that are silenced by aberrant methylation in renal cell carcinoma (RCC), we subjected two RCC lines to methylated CpG island amplification/representational difference analysis. This identified 27 CpG islands. Combined bisulfite restriction analysis of these CpG islands in primary RCC cases revealed that four were methylated in a tumor-specific manner. One of these was identified as the human homeo-box gene B13 (HOXB13) gene, but the remaining three CpG islands were not associated with known genes. The methylation frequencies of HOXB13 in primary RCC samples and lines were 30 and 73%, respectively. The methylation status of HOXB13 correlated with the loss of its expression both in RCC lines and primary tumors, and methyltransferase inhibitor treatment induced the recovery of its expression. Exogenous expression of HOXB13 in RCC cells that lacked endogenous HOXB13 expression suppressed colony formation and induced apoptotic features. Furthermore, HOXB13 methylation correlated positively with tumor grade and microvessel invasion. These results suggest that HOXB13 is a novel candidate tumor suppressor gene in RCC and that its inactivation may play an important role in both RCC tumorigenesis and progression.

Promoter Hypermethylation of the Bone Morphogenetic Protein-6 Gene in Malignant Lymphoma

Purpose: Bone morphogenetic proteins (BMP), belonging to the transforming growth factor-β superfamily, are important regulators of cell growth, differentiation, and apoptosis. The biological effects of BMPs on malignant lymphoma, however, remain unknown. Promoter methylation of the BMP-6 gene in lymphomas was investigated. Experimental Design: We investigated BMP-6 promoter methylation and its gene expression in various histologic types of 90 primary lymphomas and 30 lymphoma cell lines. The effect of BMP-6 promoter hypermethylation on clinical outcome was also evaluated. Results:BMP-6 was epigenetically inactivated in subsets of lymphomas. The silencing occurred with high frequency in diffuse large B-cell lymphoma (DLBCL) and Burkitts lymphoma in association with aberrant BMP-6 promoter methylation. The methylation was observed in 60% (21 of 35) of DLBCL cases and 100% (7 of 7) of DLBCL cell lines, and in 83% (5 of 6) of Burkitts lymphoma cases and 86% (12 of 14) of Burkitts lymphoma cell lines. In contrast, other histologic types of primary lymphomas studied had little or no detectable methylation (1 of 49; 2%). The presence of BMP-6 promoter hypermethylation in DLBCL statistically correlated with a decrease in disease-free survival (P = 0.014) and overall survival (P = 0.038). Multivariate analysis showed that the methylation profile was an independent prognostic factor in predicting disease-free survival (P = 0.022) and overall survival (P = 0. 046). Conclusion:BMP-6 promoter was hypermethylated more often in aggressive types of lymphomas, and the hypermethylation is likely to be related to the histologic type of lymphomas. BMP-6 promoter methylation may be a potential new biomarker of risk prediction in DLBCL.

Emergency Pancreatoduodenectomy for Pancreatic Metastasis from Renal Cell Carcinoma in a Patient with von Hipple-Lindau Disease: A Case Report

Von Hippel-Lindau (VHL) disease is an autosomal-dominant neoplasia syndrome, with an estimated prevalence of 1 per 36,000 live births [1]. The types of tumor seen in patients with VHL disease include hemangioblastoma of the central nervous system and retina, renal cell carcinoma (RCC), neuroendocrine tumor of the pancreas, and pheochromocytoma of the adrenal gland. The mechanism responsible for carcinogenesis in this disease complex is a loss of tumor suppressor function resulting from a causative mutation in the VHL gene. Unusually, a patient with VHL disease inherits heterozygous mutation in the VHL gene. In such individuals, a tumor arises when the wild-type allele is inactivated by somatic mutation in a susceptible organ (Knudson’s two-

Ternary complex formation of pVHL, elongin B and elongin C visualized in living cells by a fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique.

The tumor suppressor von Hippel–Lindau (VHL) gene product forms a complex with elongin B and elongin C, and acts as a recognition subunit of a ubiquitin E3 ligase. Interactions between components in the complex were investigated in living cells by fluorescence resonance energy transfer (FRET)–fluorescence lifetime imaging microscopy (FLIM). Elongin B–cerulean or cerulean–elongin B was coexpressed with elongin C‐citrine or citrine‐elongin C in CHO‐K1 cells. FRET signals were examined by measuring a change in the fluorescence lifetime of donors and by monitoring a corresponding fluorescence rise of acceptors. Clear FRET signals between elongin B and elongin C were observed in all combinations, except for the combination of elongin B‐cerulean and citrine‐elongin C. Although similar experiments to examine interaction between pVHL30 and elongin C linked to cerulean or citrine were performed, FRET signals were rarely observed among all the combinations. However, the signal was greatly increased by coexpression of elongin B. These results, together with results of coimmunoprecipitation experiment using pVHL, elongin C and elongin B, suggest that a conformational change of elongin C and/or pVHL was induced by binding of elongin B. The conformational change of elongin C was investigated by measuring changes in the intramolecular FRET signal of elongin C linked to cerulean and citrine at its N‐ and C‐terminus, respectively. A strong FRET signal was observed in the absence of elongin B, and this signal was modestly increased by coexpression of elongin B, demonstrating that a conformation change of elongin C was induced by the binding of elongin B.