関 直彦

To investigate the hepatitis B virus (HBV) genotype-related differences in the progression of liver disease, 585 patients with chronic HBV infection including 258 with histologically verified chronic liver disease (CLD) and 74 with hepatocellular carcinoma (HCC) were examined. The mean ages of both patients with advanced fibrosis (F3 or F4) and with HCC were significantly older in genotype B than in genotype C patients (P =.018, P =.024, respectively). Both the hepatitis B e antigen (HBeAg) negativity rate at biopsy and the cumulative HBe seroconversion rate in patients with CLD were significantly higher in genotype B patients than genotype C patients (P <.01, P =.022, respectively). Multivariate analysis revealed that genotype B, presence of precore mutation, high ALT levels, and severe histologic activity were independent factors for HBe seroconversion. Among all the biopsy-proven CLD patients, the ratio of patients with advanced fibrosis in genotype B was significantly lower than that in genotype C (4/30 vs. 74/224, respectively; P =.034). This difference was more remarkable in younger patients (≤45 years; 1/25 vs. 47/180, respectively; P =.020), and there was no difference in older patients (> 45 years). The distribution of each genotype between CLD and HCC was very similar (B and C: 11.2% and 87.0% vs. 10.8% and 89.2%, respectively). In conclusion, our results suggest that, although the patients with genotype B experience earlier HBe seroconversion, slower progression of liver fibrosis, and slower development of HCC, the life-long risk of progression to advanced fibrosis and development of HCC may not differ among genotypes B- and C-related chronic liver disease.

Although hepatitis C virus (HCV) is a causative agent of liver diseases, its mechanism of pathogenesis is still unclear, mainly because of the lack of adequate cell culture systems to support HCV infection and replication. In this report, we describe development and characterization of human hepatoma cell lines constitutively expressing entire (Hep394) or parts (Hep352, Hep3294) of the HCV open reading frame (ORF). The viral and cellular proteolytic machinery involved in the viral precursor processing was consistently functional, and processed HCV proteins were synthesized in these established cell lines. By using a cDNA microarray analysis coupled with semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), we identified 12 genes up-regulated and 4 genes down-regulated in Hep394 cells. With regard to genes related to cell growth regulation, we found up-regulation of forkhead transcription factor FREAC-1, poly (A) binding protein PABP2, and Ras suppressor Rsu-1. Another category of changes in gene expression includes MHC antigens, which play an important role in the T-cell-mediated immune reaction in the liver. In conclusion, functional genomic approaches comparing expression among the different cell lines expressing parts of the HCV genome may promote our understanding of the molecular basis of pathogenicity of HCV infection.

Objective-Src homology 2-containing phosphotyrosine phosphatase 2 (SHP2) is ubiquitously expressed and believed to function as part of a positive signaling pathway mediating growth factor-induced protein tyrosine phosphorylation. Proliferation of aortic vascular smooth muscle cells (SMCs) is an important contributor to atherosclerosis. We examined the effect. of SHP2 expression on SMC proliferative activity. Methods and Results-SHP2 was abundant in cultured aortic SMCs, and SHP2 staining was markedly increased in the thickened aortic intima in rats., with balloon-induced injury. We. obtained several SMC clones by using geneticin screening. Endogenous SHP2 expression varied: among individual clones. Significant positive relationships were observed. between SHP2 expression and bromodeoxyuridine uptake in SMCs stimulated by FBS, platelet-derived growth or insulin-like growth factor-1. In SMCs transiently transfected With SHP2, FBS stimulation significantly increased bromodeoxypridine. uptake beyond the uptake. by control,SMCs. Conclusions-Increased,SHP2 expression SMCs may accelerate aortic atherosclerosis by increasing cell growth.

Background. MASH1, a transcription factor with basic helix-loop-helix domain, has a pivotal function to promote differentiation of neural crest cells into autonomic neurons. Procedure. To investigate the functional significance of human MASH1 (hASH1) in the pathogenesis of neuroblastoma, which is originated from autonomic precursor cells, we studied hASH1 gene expression in primary neuroblastomas and human neuroblastoma cell lines. Results. The following results were obtained: (i) hASH1 was expressed in 40 out of 61 (66%) primary neuroblastomas, (ii) hASH1 transcripts were downregulated in several cell lines prior to differentiation induced by all-trans retinoic acid (RA), (iii) a neuroblastoma cell line without expression of endogenous hASH1 did not respond to RA at all, and (iv) the analysis of the hASH1 genomic DNA revealed two possible transcription initiation sites, which may correspond to 3.0 kb and 3.5 kb transcripts. Conclusions. Our observations suggest that, although hASH1 may not preserve the growth capacity of neuroblastomas, downregulation of hASH1 may be necessary to promote neuronal differentiation of neuroblastoma. Med. Pediatr. Oncol. 36:132-134, 2001. (C) 2001 Wiley-Liss. Inc.

Background. Human p73, a novel homolog of p53, has recently been cloned and mapped at chromosome 1p36.3, the locus for putative tumor suppressor gene(s) of neuroblastoma (NBL) and other cancers, p73, like p53, inhibits growth and induces apoptosis in neuroblastoma and osteosarcoma cell lines. Procedure. To lest the hypothesis that p73 is a NBL suppressor gene, we examined expression, alleto-typing, and mutation of the p73 gene in primary human neuroblastomas. Loss of heterozygosity (LOH) for p73 was performed in 272 primary NBLs using a CT repeat polymorphic marker, which eve found in intron 9 of the p73 gene. Results. p73 LOH was observed in 28 out of 151 (19%) informative cases. The high frequency of p73 LOH was significantly associated with sporadic neuroblastomas (P < 0.001), MYCN amplification (P < 0.001), and advanced stages (P < 0.05). Mutational analyses by PCR-SSCP (single strand conformation polymorphism) revealed two mis-sense mutations in 140 NBLs, one somatic and one germline. Conclusion. Thus, the present results have shown that mutation of p73 is infrequent in NBLs, although the p73 locus is frequently lost in advanced stage rumors. These suggest that p73 may not be a tumor suppressor in the classic Knudson manner. Med. Pediatr. Oncol. 36:42-44, 2001. (C) 2001 Wiley-Liss. Inc.

The BRCT (<(BR)under bar>ECA1 (C) under bar-(t) under bar erminus) superfamily includes a large number of nuclear proteins closely involved in DNA repair, recombination, and cell-cycle control. The human cDNA clone NFBD1 (previously designated KIAA0170) encodes a novel protein (2089 amino acids in length; calculated molecular mass 226,440 D) with possible BRCT domains at its carboxy terminus (amino acid residues 1894-2089), This gene product has been described as one of the BRCT superfamily proteins. However, its biological significance has been unclarified, Expression of green fluorescent protein (GFP)-tagged full-length NFBD1 or a series of deletion mutants indicated that NFBD1 was localized to the nucleus in various mammalian cells, and a 197-amino acid segment near the amino terminus (amino acid residues 142-338) contained a nuclear targeting signal. In vitro DNA-binding experiments showed that the highly basic region of NFBD1 (amino acid residues 1841-1893) possessed DNA-binding activity. The region encoding amino acids 508-995 of NFBD1 fused inframe with GAL4 DNA-binding domain activated transcription in both yeast and mammalian cells, while the possible BRCT domains of NFBD1 failed to induce transcription in mammalian cells. Overexpression of antisense NFBD1 RNA in a p53-deficient human osteogenic sarcoma cell line (SAOS-2) resulted in remarkable suppression of SAOS-2 colony formation. These results suggest that NFBD1 is a nuclear transcriptional transactivator with possible BRCT domains and may contribute to cell growth control.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-40 alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4 alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T130l in exon 4: and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T130l in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4 alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4 alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.

The distal region of a short arm of chromosome 1p is frequently deleted in many human cancers including neuroblastoma (NBL), in which it has been narrowed down to the smallest region of overlap between D1S244 and D1S214 (approximately 7 cM). During the search for the candidate tumor suppressor genes mapped within the region, we found the KIAA0591 gene which encoded a new human kinesin-related protein with a homology to human axonal transporter of synaptic vesicles (ATSV). The kinesin is an intracellular motor protein and often associated with neuronal differentiation and survival. Here we identified a complete open reading frame of the KIAA0591 gene by screening a cDNA library derived from human substantia nigra. The KIAA0591 protein contains a possible pleckstrin homology (PH) domain at its carboxy-terminus. However, it did not possess a force-generating motor domain which is well conserved among kinesin superfamily members (KIFs). Northern blot analysis demonstrated that KIAA0591 mRNA was preferentially expressed in both adult and fetal brains, kidney, skeletal muscle and pancreas. KIAA0591 was expressed in favorable NBLs at higher levels than in unfavorable NBLs, although RT-PCR SSCP analysis showed no mutation within the coding region of the KIAA0591 gene, when 8 neuroblastoma tissues and 15 neuroblastoma-derived cell lines were examined. Thus, the full-length KIAA0591 gene may be a novel member of human KIF superfamily which lacks motor domain and might function as a tumor suppressor in an epigenetic but not a classic Knudson's manner.